Amylin family peptides and methods for making and using them

ABSTRACT

The present invention relates to novel compounds having a function of a peptide in the amylin family, related nucleic acids, expression constructs, host cells, and processes production of the compounds. The compounds of the invention include one or more amino acid sequence modifications. In addition, methods and compositions are disclosed to treat and prevent metabolic disorders such as obesity, diabetes, and increased cardiovascular risk.

RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.10/589,054, filed Aug. 10, 2006, now U.S. Pat. No. 8,114,958 issued Feb.14, 2012, which is a national stage filing of International ApplicationNo. PCT/US2005/004631, filed Feb. 11, 2005, which claims the benefit ofpriority under 35 U.S.C. §119(e) of U.S. Provisional Application No.60/543,275, filed Feb. 11, 2004, and of U.S. Provisional Application No.60/550,447, filed Mar. 3, 2004, all of which are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 31, 2012, isnamed 0105US-DIV2-SEQ_ST25_(—)2011-Mar.txt and is 70,209 bytes in size.

TECHNICAL FIELD

The present invention relates to novel compounds and methods for makingand using them. These compounds may be useful for treating or preventingconditions such as metabolic disorders, vascular disorders, renaldisorders, and/or gastrointestinal disorders. An exemplary conditionmight be one in which the reduction of food intake or caloric intake isof value, e.g., obesity, Type II diabetes, eating disorders, metabolicsyndrome and insulin-resistance syndrome.

BACKGROUND

Much work had been done to date on amylin, calcitonin, and calcitoningene related peptide (CGRP) to understand their structure and function.Table 1 provides a summary of some biological effects of CGRP,calcitonin and amylin as published in Wimalawansa, S. J. (1997) CriticalReviews in Neurobiology, 11; 167-239.

TABLE 1 Biological effect CGRP Calcitonin Amylin Vasodilation ++++ +/−++ Chronotropic ++ − ++ Inotropic ++ − ++ Inhibit bone resorption + ++++++ Stimulate bone formation − − + Calcium-lowering effect − +++ ++Growth factor-like effect + − + Neural regeneration ++ +/− −Thermoregulation + − + Inflammation ++ +/− − Gastric mucosal protection++ − − Effects on β cells + +/− ++ Skeletal muscle + − + Glucosemetabolism + − + Anorectic effect + ++ ++ Analgesic effect − ++ +

Amylin has been reported to regulate gastric emptying and suppressglucagon secretion and food intake, thus regulating the rate of glucoseappearance in the circulation. It appears to complement the actions ofinsulin, which regulates the rate of glucose disappearance from thecirculation and its uptake by peripheral tissues. These actions aresupported by experimental findings in rodents and humans, which indicatethat amylin complements the effects of insulin in postprandial glucosecontrol by at least three independent mechanisms, all of which affectthe rate of glucose appearance. First, amylin suppresses postprandialglucagon secretion. Compared to healthy adults, patients with type 1diabetes have no circulating amylin and patients with type 2 diabeteshave diminished postprandial amylin concentrations. Furthermore,infusion of an amylin specific monoclonal antibody, which boundcirculating amylin, again resulted in greatly elevated glucagonconcentrations relative to controls. Both of these results point to aphysiological role of endogenous amylin in the regulation ofpostprandial glucagon secretion. Second, amylin slows gastrointestinalmotility and gastric emptying. Finally, intrahypothalamic injections ofrat amylin were shown to reduce feeding in rats and alterneurotransmitter metabolism in the hypothalamus. In certain studies,food intake was significantly reduced for up to eight hours followingthe intrahypothalamic injection of rat amylin and rat CGRP. In humantrials, an amylin analog, pramlintide, has been shown to reduce weightor weight gain. Amylin may be beneficial in treating metabolicconditions such as diabetes and obesity. Amylin may also be used totreat pain, bone disorders, gastritis, to modulate lipids, in particulartriglycerides, or to affect body composition such as the preferentialloss of fat and sparing of lean tissue.

The hormone calcitonin (CT) was named for its secretion in response toinduced hypercalcemia and its rapid hypocalcemic effect. It is producedin and secreted from neuroendocrine cells in the thyroid that have sincebeen termed C cells. The best-studied action of CT(1-32) is its effecton the osteoclast. In vitro effects of CT include the rapid loss ofruffled borders and decreased release of lysosomal enzymes. Ultimately,the inhibition of osteoclast functions by CT results in a decrease inbone resorption. However, neither a chronic reduction of serum CT in thecase of thyroidectomy nor the increased serum CT found in medullarythyroid cancer appears to be associated with changes in serum calcium orbone mass. It is thus most likely that a major function of CT(1-32) isto combat acute hypercalcemia in emergency situations and/or protect theskeleton during periods of “calcium stress” such as growth, pregnancy,and lactation. (Reviewed in Becker, JCEM, 89(4): 1512-1525 (2004) andSexton, Current Medicinal Chemistry 6: 1067-1093 (1999)). Consistentwith this is recent data from the calcitonin gene knockout mouse, whichremoves both the calcitonin and the CGRP-I peptides, that revealed thatthe mouse had normal levels of basal calcium-related values, but anincreased calcemic response (Kurihara H, et al, Hypertens Res. 2003February; 26 Suppl:S 105-8).

CT has an effect on plasma calcium levels and inhibits osteoclastfunction and is widely used for the treatment of osteoporosis.Therapeutically, salmon CT appears to increase bone density and decreasefracture rates with minimal adverse effects. CT has also beensuccessfully used over the past 25 years as a therapy for Paget'sdisease of bone, which is a chronic skeletal disorder that may result inenlarged or deformed bones in one or more regions of the skeleton. CT isalso widely used for its analgesic effect on bone pain experiencedduring osteoporosis, although the mechanism for this effect is notclearly understood.

Calcitonin gene related peptide (CGRP) is a neuropeptide whose receptorsare widely distributed in the body, including the nervous system and thecardiovascular system. This peptide seems to modulate sensoryneurotransmission and is one of the most potent endogenous vasodilatorypeptide discovered to date. Reported biological effects for CGRPinclude: modulation of substance P in inflammation, nicotinic receptoractivity at the neuromuscular junction, stimulation of pancreatic enzymesecretion, a reduction of gastric acid secretion, peripheralvasodilation, cardiac acceleration, neuro-modulation, regulation ofcalcium metabolism, osteogenic stimulation, insulin secretion, anincrease in body temperature and a decrease in food intake.(Wimalawansa, Amylin, calcitonin gene-related peptide, calcitonin andADM: a peptide superfamily. Crit Rev Neurobiol. 1997; 11(2-3): 167-239).An important role of CGRP is to control blood flow to various organs byits potent vasodilatory actions, as evidenced by a decrease of meanarterial pressure following intravenous administration of CGRP. Thevasodilatory actions are also supported by recent analysis of homozygousknockout CGRP mice, which demonstrated elevated peripheral vascularresistance and high blood pressure caused by increased peripheralsympathetic activity (Kurihara H, et al, Targeted disruption of ADM andalphaCGRP genes reveals their distinct biological roles. Hypertens Res.2003 February; 26 SuppliS105-8). Thus, CGRP appears to elicitvasodilatory effects, hypotensive effects and an increase in heart rateamong other actions.

Prolonged infusion of CGRP into patients with congestive cardiac failurehas shown a sustained beneficial effect on hemodynamic functions withoutadverse effects, suggesting a use in heart failure. Other indications ofCGRP use include renal failure, acute and chronic coronary arteryischemia, treatment of cardiac arrhythmia, other peripheral vasculardisease such as Raynaud's phenomenon, subarachnoid hemorrhage,hypertension, and pulmonary hypertension. Preeclamptic toxemia ofpregnancy and preterm labor is also potentially treatable. (Wimalawansa,1997). Recent therapeutic uses include the use of CGRP antagonists forthe treatment of migraine headaches.

There are many beneficial properties of each peptide that can be usedalone or in combination to treat or prevent a variety of conditions. Itwould be useful to create new and useful peptides having multipleactions that impart improved characteristics not possessed by theexisting peptides. For example, in food intake assays, amylin has beenshown to have a quick onset, within 30 minutes, but its effect tapersoff after 60 minutes. In contrast, salmon calcitonin has been shown tohave a delayed effect, with peak levels still maintained at 240 minutes.Novel compounds that can mimic the effects of amylin and/or calcitoninand have quick onset of activity like amylin with the sustained activityof calcitonin may increase the potency and efficacy of either compoundalone. Moreover, the combination of certain physicochemicalcharacteristics of amylin, calcitonin, and/or CGRP into a singlemodality may facilitate intervention at different points in adysfunctional metabolic circuit. These novel compounds combine desirableactivities or properties for a superior therapeutic, which may result incompounds having at least one desirable characteristic such as higherefficacy, greater potency, greater bioavailability, fewer side effects,ease in manufacture, stability, and/or solubility.

All documents referred to herein are incorporated by reference into thepresent application as though fully set forth herein.

SUMMARY

The present invention relates, at least in part, to novel amylin familypeptides or compound. In a general aspect, these novel compounds (alsoreferred to as compounds of the invention) have at least a loop regionof amylin or calcitonin and analogs thereof; an α helix region of atleast a portion of an α helix region of calcitonin or analogs thereof oran α helix region having a portion of an amylin α helix region and acalcitonin α helix region, or their respective analogs; and a C-terminaltail of amylin or calcitonin or analogs thereof, with the proviso thatthe C-terminal tail of calcitonin or a calcitonin analog is not proline(Pro), hydroxyproline (Hyp), homoSerine (Hse) or derivatives of Hse.

As used herein, an “analog” refers to a peptide whose sequence wasderived from that of a base reference peptide, e.g., amylin andcalcitonin, and includes insertions, substitutions, extensions, and/ordeletions of the reference amino acid sequence, preferably having atleast 50 or 55% amino acid sequence identity with the base peptide, morepreferably having at least 70%, 80%, 90%, or 95% amino acid sequenceidentity with the base peptide. In one embodiment, such analogs maycomprise conservative or non-conservative amino acid substitutions(including non-natural amino acids and L and D forms). Analogs includecompounds having agonist and compounds having antagonist activity.Analogs, as herein defined, also include derivatives.

A “derivative” is defined as a reference peptide or analogs, describedabove, having a chemical modification of one or more of its amino acidside groups, α-carbon atoms, terminal amino group, or terminalcarboxylic acid group. A chemical modification includes, but is notlimited to, adding chemical moieties, creating new bonds, and removingchemical moieties. Modifications at amino acid side groups include,without limitation, acylation of lysine ε-amino groups, N-alkylation ofarginine, histidine, or lysine, alkylation of glutamic or asparticcarboxylic acid groups, and deamidation of glutamine or asparagine.Modifications of the terminal amino include, without limitation, thedesamino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications.Modifications of the terminal amino include, without limitation, thedesamino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications,such as alkyl acyls, branched alkylacyls, alkylaryl-acyls. Modificationsof the terminal carboxy group include, without limitation, the amide,lower alkyl amide, dialkyl amide, arylamide, alkylarylamide and loweralkyl ester modifications. Lower alkyl is C1-C4 alkyl. Furthermore, oneor more side groups, or terminal groups, may be protected by protectivegroups known to the ordinarily-skilled synthetic chemist. The α-carbonof an amino acid may be mono- or dimethylated.

In certain embodiments, the novel compounds have an amylin or amylinanalog loop region, at least a portion of a calcitonin or calcitoninanalog α helix region, and an amylin or amylin analog C-terminal tail.In other embodiments, the novel compounds have a calcitonin orcalcitonin analog loop region, at least a portion of a calcitonin orcalcitonin analog α helix region, and an amylin or amylin analogC-terminal tail. In still other embodiments, the novel compounds have anamylin or amylin analog loop region, at least a portion of an amylin oramylin analog α helix region and at least a portion of a calcitonin orcalcitonin analog α helix region, and an amylin or amylin analogC-terminal tail. In yet other embodiments, the novel compounds have acalcitonin or calcitonin analog loop region, at least a portion of anamylin or amylin analog α helix region and at least a portion of acalcitonin or calcitonin analog α helix region, and an amylin or amylinanalog C-terminal tail. In still yet other embodiments, the novelcompounds have an amylin or amylin analog loop region, a portion or acalcitonin or calcitonin analog α helix region or at least a portion ofan amylin or amylin analog α helix region and at least a portion of acalcitonin or calcitonin analog α helix region, and a calcitonin orcalcitonin analog C-terminal tail.

In certain embodiments, the loop region of the novel compounds mayfurther comprise no more than one, two, three, or four modificationsincluding substitutions, insertions, or deletions from the amylin orcalcitonin loop, and analogs thereof. It is further contemplated thatthe novel compounds may have additional modifications at the N-terminalportion of the loop comprising a N-cap region, that may have hydrophobicor hydrophilic characteristics such as acetyl, isocaproyl,3,6-dioxyoctanoic acid, or 1-amino-4,7,10-trioxa-13-tridecanaminesuccinimic acid. Modifications may further include one, two, three ormore additional amino acids. This is an area which allows for manymodifications too numerous to mention, but would be understood by one ofskill in the art based upon what is exemplified further in the presentapplication.

In general, with respect to an amino acid sequence, the term“modification” includes substitutions, insertions, elongationsdeletions, and derivatizations alone or in combination. The novelcompounds of the invention may include one or more modifications of a“non-essential” amino acid residue. In the context of the invention, a“non-essential” amino acid residue is a residue that can be altered,i.e., deleted or substituted, in the novel amino acid sequence withoutabolishing or substantially reducing the agonist activity of the analogpolypeptide.

Substitutions include conservative amino acid substitutions. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain, or physicochemical characteristics (e.g., electrostatic, hydrogenbonding, isosteric, hydrophobic features). The amino acids may benaturally occurring or nonnatural (unnatural). Families of amino acidresidues having similar side chains are known in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, methionine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,tryptophan), β-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Substitutions may also include non-conservativechanges.

The compounds of the invention may also be further derivatized bychemical alterations such as amidation, glycosylation, acylation,sulfation, phosphorylation, acetylation, and cyclization. Such chemicalalterations may be obtained through chemical or biochemicalmethodologies, as well as through in-vivo processes, or any combinationthereof. Derivatives of the compounds of the invention may also includeconjugation to one or more polymers or small molecule substituents. Onetype of polymer conjugation is linkage or attachment of polyethyleneglycol (“PEG”) polymers, polyamino acids (e.g., poly-his, poly-arg,poly-lys, etc.) and/or fatty acid chains of various lengths to the N- orC-terminus or amino acid residue side chains of an AFP-6 analog. Smallmolecule substituents include short alkyls and constrained alkyls (e.g.,branched, cyclic, fused, adamantyl), and aromatic groups. In addition,basic residues such as R and K may be replaced with homoR and homoK,citrulline, or ornithine to improve metabolic stability of the peptide.Compounds of the invention also include acid as well as amide forms ofthe peptides.

In certain embodiments, the α helix region of the novel compoundscomprise at least four consecutive amino acids of a calcitonin orcalcitonin analog α helix region. In other embodiment, the α helixregion comprises at least 5, 6, 7, or 8 consecutive amino acids of acalcitonin or calcitonin analog α helix region. In other embodiments,the α helix region comprises at least 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21 or more consecutive amino acids of a calcitonin orcalcitonin analog α helix region. In certain embodiments, when thenumber of consecutive amino acids are less than 8, it is contemplatedthat the α helix region further comprises at least 4, 5, 6, 7, 9, 10,11, or more consecutive amino acid of an amylin or amylin analog α helixregion. In certain embodiments, it is envisioned that the less aminoacids of calcitonin or calcitonin analog, the more amino acids of anamylin or amylin analog may be found in the α helix region of the novelcompounds. The number of amino acids comprising the α helix region maybe from about 10 to 23 amino acids. Accordingly, the α helix region maybe 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acidslong. Moreover, the amino acids should provide for about three to aboutsix α helical turns. It is further contemplated that the α helix regionof the novel compounds may further comprise no more than one, two,three, four, five, six, seven, eight or 10 modifications includingsubstitutions, insertions, or deletions from that of the calcitoninand/or amylin α helix region, and analogs thereof.

In certain embodiments, the C-terminal tail of the novel compoundscomprise at least the last six, five, or four amino acids of eitheramylin or calcitonin, and analogs thereof. In certain embodiments, theC-terminal tail of the novel compounds comprise at least a portion ofthe C-terminal end having a β turn. In certain embodiments, the β turnis introduced by the amino acid combination of Gly-Ser. Accordingly, thenovel compounds may have a C-terminal end comprising a portion of anamylin or calcitonin C-terminal tail (and analogs thereof) havingGly-Ser or starting at Gly-Ser.

In certain embodiments, the C-terminal tail of the novel compounds mayfurther comprise no more than one, two, or three, modificationsincluding substitutions, insertions, or deletions from the amylin orcalcitonin loop, and analogs thereof. It is further contemplated thatthe novel compounds may have additional modifications at the C-terminalportion of the C-terminal tail which may include L-octylglycine, 4ABU(4-aminobutyric acid), 9Anc (9 amiononanoic acid, 3,6-dioxyoctanoic acidor 1-amino-4,7,10-trioxa-13-tridecanamine succinimic acid. Modificationmay further include one, two, three or more additional amino acids. Thetypes of modification contemplated in this area would be understood byone of skill in the art based upon what is exemplified further in thepresent application.

In one aspect, a loop region is defined as that region found at theN-terminal end comprising at least 5 to 8 amino acids, wherein the firstand last amino acid are capable of creating a bond, for example,residues at positions 2-7 of amylin or residues at positions 1-7 ofcalcitonin and their corresponding regions in their respective analogs.In another aspect, a α helix region is defined as the internal portionof amylin or calcitonin flanked by the loop region and the C-terminaltail which structurally forms an α helix, for example, residues atpositions 8-23 of amylin or residues at positions 8-27 of calcitonin andtheir corresponding regions in their respective analogs. In yet anotheraspect, a C-terminal tail is defined as that region after the α helixe.g., residues at positions 33-37 of amylin or longer such as residuesat positions 27-37 or residues at positions 27 or 28 to 32 ofcalcitonin. Included in the compounds of the invention are both theamide and acid forms of the disclosed compounds.

Amylin and calcitonin, as herein defined, includes all native andspecies variations. Examples of amylin and calcitonin include, but arenot limited to:

human amylin (hAmylin) (SEQ ID NO: 1)KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY rat amylin (rAmylin)(SEQ ID NO: 2) KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTYsalmon calcitonin (sCT) (SEQ ID NO: 3) CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTPhuman calcitonin (hCT) (SEQ ID NO: 4) CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP.

By “amino acid” and “amino acid residue” is meant natural amino acids,unnatural amino acids, and modified amino acid. Unless stated to thecontrary, any reference to an amino acid, generally or specifically byname, includes reference to both the D and the L stereoisomers if theirstructure allow such stereoisomeric forms. Natural amino acids includealanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp),cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly),histidine (His), isoleucine (Ile), leucine (Leu), Lysine (Lys),methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).Unnatural amino acids include, but are not limited to homolysine,homoarginine, homoserine azetidinecarboxylic acid, 2-aminoadipic acid,3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyricacid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid,2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid,tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline.Additional unnatural amino acids include modified amino acid residueswhich are chemically blocked, reversibly or irreversibly, or chemicallymodified on their N-terminal amino group or their side chain groups, asfor example, N-methylated D and L amino acids or residues wherein theside chain functional groups are chemically modified to anotherfunctional group. For example, modified amino acids include methioninesulfoxide; methionine sulfone; aspartic acid-(beta-methyl ester), amodified amino acid of aspartic acid; N-ethylglycine, a modified aminoacid of glycine; or alanine carboxamide, a modified amino acid ofalanine. Additional residues that can be incorporated are described inSandberg et al., J. Med. Chem. 41: 2481-91, 1998.

In a general aspect, compounds of the invention comprise at least a loopregion, an α helix region, and a C-terminal tail. The loop regioncomprises an amino sequence comprising the formula X-Xaa1 sequence-Ywherein X and Y are capable of creating a bond and are independentlyselected residues having side chains which are chemically bonded to eachother to form an intramolecular linkage such as disulfide bonds; amidebond; alkyl acids and alkyl amines which may form cyclic lactams; alkylaldehydes or alkyl halides and alkylamines which may condensed and bereduced to form an alkyl amine or imine bridge; or side chains which maybe connected to form an alkyl, alkenyl, alkynyl, ether or thioetherbond. Alkyl chains may include lower alkyl groups having from about 1 toabout 6 carbon atoms. In certain embodiments, the intramolecular linkagemay be a disulfide, amide, imine, amine, alkyl and alkene bond. Incertain embodiments, X and Y are independently selected from Ser, Asp,Glu, Lys, Orn, or Cys. In certain embodiments, X and Y are Cys and Cys.In other embodiments, X and Y are Ser and Ser. In still otherembodiments, X and Y are Asp and Lys or Lys and Asp.

The Xaa1 sequence comprises an amino acid sequence of 3, 4, 5, or 6amino acids between X and Y. In certain embodiments, the Xaa1 sequencecomprises an amino acid sequence having a region with one or moresubstituted or unsubstituted hydroxyl-containing residues next to Y. Forexample, the hydroxyl containing residue region may have at least 2 ofthe 3 amino acids adjacent Y that are either a Ser or Thr. The otheramino acids in the Xaa1 sequence may be any amino acid. In certainembodiments, the Xaa1 sequence is 3 amino acids. In other embodiments,the Xaa1 sequence is 4 amino acids. In still other embodiments, the Xaa1sequence is 5 amino acids. In yet other embodiments, the Xaa1 sequenceis 6 amino acids. Accordingly, Xaa1 can be represented byXaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7 (SEQ ID NO:5). In certain embodiments,Xaa2, Xaa3, and/or Xaa4 may absent. In certain embodiments, Xaa5, Xaa6,and Xaa7 comprise the hydroxy-containing residue region. As such, atleast two of the three amino acids can be a Ser, hSer, Thr, alloThr,d-Thr, or other unnatural analog thereof. Xaa2 can be any amino acid orabsent, Xaa3 can be any amino acid or absent, Xaa4 can be any amino acidor absent, Xaa5 can be any amino acid if Xaa6 is a Ser or Thr and Xaa7is a Ser or Thr, Xaa6 can be any amino acid if Xaa5 is a Ser or Thr andXaa7 is a Ser or Thr, Xaa7 can be any amino acid if Xaa5 is Ser or Thrand Xaa6 is Ser or Thr. Accordingly, in certain embodiment, Xaa1 can berepresented as Xaa2 absent, Xaa3 is Ala, Gly, Ser, Asp or absent, Xaa4is Asn, Ala, Asp, Gly or absent; Xaa5 is Ala, Leu, Thr, or Ser; Xaa6 isAla, Ser, or Thr; and Xaa7 is Ala, Ser, Val, Hse,(S)-2-amio-3-hydroxy-methylbutanoic acid (Ahb),(2S,3R)-2-amino-3hydroxy-methylpentanoic acid (Ahp), D-Thr, Thr, or aderivative thereof. In other embodiments Xaa1 can be represented as Xaa2is absent, Xaa3 is Ser, Gly, or absent, Xaa4 is Asn or Asp, Xaa5 is Ala,Ser, Thr or Leu, Xaa6 is Ala, Thr or Ser, and Xaa7 is Ser, D-Thr,alloThr or Thr. In certain embodiments, the loop region comprises theabove-described representations of Xaa1 wherein Xaa3 is Ala, whereinXaa3 is Ser or wherein Xaa3 is Gly. Alternatively or additionally, theloop region comprises the above described representations of Xaa1wherein Xaa4 is Ala, wherein Xaa4 is Asn, wherein Xaa4 is Asp, orwherein Xaa4 is Gly. Alternatively or additionally, the loop regioncomprises the above-described representations of Xaa1 wherein Xaa5 isAla, wherein Xaa5 is Thr, or wherein Xaa5 is Leu. Alternatively oradditionally, the loop region comprises the above describedrepresentations of Xaa1 wherein Xaa6 is Ser or wherein Xaa6 is Ala.Alternatively or additionally, the loop region comprises theabove-described representations of Xaa1 wherein Xaa7 is Thr or whereinXaa7 is D-Thr. It is further contemplated that no more than one, two, orthree modifications such as substitutions, insertions, deletions, and/orderivatizations may be made to the loop region.

It should be noted that throughout the application that alternatives arewritten in Markush groups, for example, each amino acid position thatcontains more than one possible amino acid. It is specificallycontemplated that each member of the Markush group should be consideredseparately, thereby comprising another embodiment of the invention, andthe Markush group is not to be read as a single unit.

Examples of the loop region of the invention include, but are notlimited to, C-N-T-A-T-C (SEQ ID NO:6); C-A-T-A-T-C (SEQ ID NO:7);C-D-T-A-T-C (SEQ ID NO:8); C-G-T-A-T-C (SEQ ID NO:9); C-N-A-A-T-C (SEQID NO:10); C-N-T-S-T-C (SEQ ID NO:11; C-N-T-A-dThr-C (SEQ ID NO:12);C-N-T-A-T(OPO3H2)-C (SEQ ID NO:13); C-N-T-A-S-C (SEQ ID NO:14);C-N-T-A-A-C (SEQ ID NO:15); C-N-T-A-V-C (SEQ ID NO:16); C-N-T-A-Hse-C(SEQ ID NO:17); C-N-T-A-Ahb-C (SEQ ID NO:18); C-N-T-A-Ahp-C (SEQ IDNO:19); C-S-N-L-S-T-C (SEQ ID NO:20); C-G-N-L-S-T-C (SEQ ID NO:21);C-A-N-L-S-T-C (SEQ ID NO:22); C-S-A-L-S-T-C (SEQ ID NO:23);C-S-N-A-S-T-C (SEQ ID NO:24); C-S-N-L-A-T-C (SEQ ID NO:25); andC-S-N-L-S-A-C (SEQ ID NO:26). As previously noted, it is furthercontemplated that no more than one, two, or three modifications such assubstitutions, insertions, deletions, and/or derivatizations may be madeto the loop region.

The loop region of the novel compounds may further comprisemodifications or additional amino acids at the N-terminal end. Suchmodifications include the addition of compounds such as Lys, Ala, Phe,Ile, Ser, Octylglycine, Isocap, Fmoc-3,6-dioxyoctanoic acid,Fmoc-1-amino-4,7,10-trioxa-13-tridecanamine succinimic acid, acetyl,and/or groups for solubility, delivery, signaling. Exemplary modifiedloops include the addition of Lys the sequence of Xaa1 or the additionof Ile to the sequence of Xaa1. For example, the modified loop regionmay be K-C-N-T-A-T-C (SEQ ID NO:27). In certain embodiments, theadditions and/or modifications at the N-terminal end of the loop regionmay change the loop region. For example, the loop region of theinvention may be modified as follows: cyclo(2,7) 1-7 hAmylin, cyclo(2Asp7Lys) 1-7 hAmylin, N-isocaproyl 1-7 hAmylin, N-3,6 dioxaoctanoyl 1-7hAmylin, L-Octylglycine 1-7 hAmylin, Acetyl (2Agy, 7Agy) 1-7 hAmylinwherein Agy is Allylglycine, Acetyl (1Ala) 1-7 hAmylin, (1Thr 3Asp) 1-7hAmylin, Isocap (7Ala) 5-7 sCT, Acetyl (2Agy, 7Agy) 1-7 sCT, and cyclo(1,7) (1Asp 7Lys) 1-7 sCT. Therefore, taking the example of Isocap(7Ala)5-7 sCT, certain embodiments of the invention comprise a modification atthe N-terminal region of the loop region of the invention such thatamino acids Xaa2 to Xaa5 are absent.

Throughout the application, the amino acid sequences may be referred toas amino acids at position a to position b adjacent to a referencepeptide. In the present application the reference peptide is amylin andcalcitonin, which sequences are provided in SEQ ID NOS:1-4. For examplein the previous paragraph, 1-7 hAmylin refers to the amino acid sequencefrom position 1 to position 7, inclusive, of human amylin (SEQ ID NO:1).Modification to the reference peptide may be shown as: position ofmodification adjacent to the modification. For example, (2Asp 7Lys) 1-7hAmylin represents the amino acid sequence at positions 1 to 7 of humanamylin with a modification of a Cys to Asp at position 2 and amodification of a Cys to Lys at position 7.

The α helix region of the novel compound may be about 8 to 23 aminoacids in length. In certain embodiments, the α helix region isamphiphatic. In certain embodiments, the α helix region comprises about3 to 6 helical turns. In certain embodiments, the α helix regioncomprises 3, 4, 5, or 6 helical turns. In other embodiments, the α helixregion is a rigid structure equivalent to about 3, 4, 5, or 6 helicalturns. An example of an idealized helix is LLQQLQKLLQKLKQY (SEQ IDNO:28). In certain embodiments, the α helix is an ampliphatic structure.Accordingly, characteristics of desirable amino acids that would providethis type of structure may be selected.

It has been found that the calcitonin α helix region, a combination ofan amylin and a calcitonin α helix region, or parts thereof, and/or someCGRP elements are desirable in the α helix region of the novelcompounds. It is contemplated that, as with the loop region, the α helixregion can be from any amylin or calcitonin, and analogs thereof.Accordingly, in certain embodiments, the α helix region is at least aportion of an α helix region of calcitonin or calcitonin analog. Inother embodiments, the α helix region is at least a portion of an αhelix region of calcitonin or calcitonin analog and at least a portionof an α helix of an amylin or amylin analog. In still other embodiments,the α helix region of the novel compounds contain elements of CGRP. Itis further contemplated that novel compounds may have no more than one,two, three, four, five, six, seven, eight, nine, or ten furthermodifications such as substitutions, insertions, deletions, and/orderivatizations.

In certain embodiments, the α helix region of the invention may compriseamino acids from position 8 of sCT to position 18, 19, 20, 21, 22, 23,24, 25, 26, or 27 of sCT. Moreover, the α helix region may comprise morethan one portion of a calcitonin or calcitonin analog α helix region ofthe same or different species, for example 8-21 sCT 19-27 sCT; 8-21 sCT18-27 sCT; or 8-16 hCT 17-27 sCT; or (11Arg) 8-16 hCT (18Arg) 17-27 sCT.Alternatively or additionally, the above described α helix of 8-18 sCTto 8-27 sCT may further comprise the substitutions of one or more of(10Aib), (11Arg), (11Orn), (11hArg), (11Cit), (11hLys), (11Lys(for)),(17Aib), (18Arg), (18Orn), (18hArg), (18Cit), (18hLys), (18Lys(for)),(18Lys(PEG5000)), (22Leu), (24Pro) or any combination thereof.

In one embodiment, an α helix region of the invention can be representedby (α helix region type I) R1-VL Xaa10 Xaa11 LSQ Xaa15 L Xaa17 Xaa18 LQTXaa22 P Xaa24 TNT-R1 (SEQ ID NO:29), wherein

Xaa10 is Gly or Aib;

Xaa11 is Lys, Arg, Orn, hArg, Cit, hLys, or Lys(for);

Xaa15 is Glu or Phe;

Xaa17 is His or Aib;

Xaa18 is Lys, Arg, Orn, hArg, Cit, hLys, Lys(for), Lys(PEG 5000);

Xaa22 is Tyr or Leu;

Xaa24 is Arg or Pro; or

R1 is absent or comprises 1-4 additional amino acids.

Again, it should be remembered that each member of the Markush group, ora combination thereof, is another embodiment of the invention and is notto be read as a single unit. This is a shorthand method for stating, asan example, embodiments of the invention include an α helix region typeI formula where, Xaa18 can be a Lys, Arg, Orn, hArg, Cit, hLys, orLys(for), and each variation is a separate embodiment of the invention.Accordingly, the α helix region type I formula has one embodiment whereXaa18 is Lys. It has another embodiment where Xaa18 is Arg, and so on.It is further contemplated that the α helix region may contain no morethan one, two, three, four, five, six, seven, eight, nine, or tenmodifications such as substitutions, insertions, deletions, and/orderivatizations. Accordingly, the compounds of α helix region type I mayhave further deletions at the C-terminal end. In certain embodiments,the amino acids of R1 are capable of forming an α helix turn.

Examples of an α helix region type I of the invention include, but arenot limited to 8-18 sCT, 8-21 sCT, 8-24 sCT, 8-27 sCT, (11Arg) 8-18 sCT,(18Arg) 8-18 sCT, (11Arg 18Arg) 8-18 sCT, (110 nm 18Orn) 8-18 sCT,(11Arg 18Cit) 8-18 sCT, (11hArg 18hArg) 8-18 sCT, (11Arg 18Orn) 8-18sCT, (11Cit 18Arg) 8-18 sCT, (11Cit 18Cit) 8-18 sCT, (11hLys 18hLys)8-18 sCT, (10Aib 11Arg 17Aib 18Arg) 8-18 sCT, (11Lys(for) 18Lys(for))8-18 sCT, (10Aib 11Lys(for) 17Aib 18Lys(for)) 8-18 sCT, (11Arg 18Lys(PEG5000)) 8-18 sCT, (11Arg) 8-21 sCT, (18Arg) 8-21 sCT, (11Arg 18Arg) 8-21sCT, (110 nm 18Orn) 8-21 sCT, (11Arg 18Cit) 8-21 sCT, (11hArg 18hArg)8-21 sCT, (11Arg 18Orn) 8-21 sCT, (11Cit 18Arg) 8-21 sCT, (11Cit 18Cit)8-21 sCT, (11hLys 18hLys) 8-21 sCT, (10Aib 11Arg 17Aib 18Arg) 8-21 sCT,(11Lys(for) 18Lys(for)) 8-21 sCT, (10Aib 11Lys(for) 17Aib 18Lys(for))8-21 sCT, (11Arg 18Lys(PEG 5000)) 8-21 sCT, (11Arg) 8-24 sCT, (18Arg)8-24 sCT, (11Arg 18Arg) 8-24 sCT, (11Arg 18Arg 22Leu) 8-24 sCT, (11Arg18Arg 24Pro) 8-24 sCT, (110 nm 18Orn) 8-24 sCT, (11Arg 18Cit) 8-24 sCT,(11hArg 18hArg) 8-24 sCT, (11Arg 18Orn) 8-24 sCT, (11Cit 18Arg) 8-24sCT, (11Cit 18Cit) 8-24 sCT, (11hLys 18hLys) 8-24 sCT, (10Aib 11Arg17Aib 18Arg) 8-24 sCT, (11Lys(for) 18Lys(for)) 8-24 sCT, (10Aib11Lys(for) 17Aib 18Lys(for)) 8-24 sCT, (11Arg 18Lys(PEG 5000)) 8-24 sCT,(11Arg) 8-27 sCT, (18Arg) 8-27 sCT, (11Arg 18Arg) 8-27 sCT, (11Arg 18Arg22Leu) 8-27 sCT, (11Arg 18Arg 24Pro) 8-27 sCT, (11Orn 18Orn) 8-27 sCT,(11Arg 18Cit) 8-27 sCT, (11hArg 18hArg) 8-27 sCT, (11Arg 18Orn) 8-27sCT, (11Cit 18Arg) 8-27 sCT, (11Cit 18Cit) 8-27 sCT, (11hLys 18hLys)8-27 sCT, (10Aib 11Arg 17Aib 18Arg) 8-27 sCT, (11Lys(for) 18Lys(for))8-27 sCT, (10Aib 11Lys(for) 17Aib 18Lys(for)) 8-27 sCT, (11Arg 18Lys(PEG5000)) 8-27 sCT, (11Arg 18Arg) 8-21 sCT-19-27 sCT, and (11Arg 18Arg)8-21 sCT-(18Leu) 18-27 sCT.

In certain embodiments, the α helix region of the invention may comprisea portion of an α helix region of amylin or amylin analog and a portionof an α helix region of calcitonin or calcitonin analog. The α helixregion of the invention may comprise amino acids from position 8 ofhAmylin to 11, 12, 13, 14, 15, 16, 17, 18 or 19 of hAmylin and aminoacids from position 13, 14, 15, 16, 17, 18, and 19 of sCT to position18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 of sCT. Alternatively oradditionally, the above described α helix region of amylin andcalcitonin may further comprise the substitutions of one or more of(8Val), (9Leu), (9Met), (10Gly), (10His), (12Thr), (13Thr), (13Asn),(13Phe), (13Tyr), (14Arg), (14Ala), (14Asp), (14Glu), (14Gln), (14Thr),(14Gly), (15Leu), (15Ser), (15Glu), (15Ala), (15Tyr), (16Asp), (17Ser),(17Phe), (18Arg), (17Aib), (18Arg), (18Orn), (18hArg), (18Cit),(18hLys), (18Lys(for)), (18Lys(PEG5000)), (19Phe), (20His), (21Asn),(22Met), (22Val), (22Phe), (22Leu), (24Pro), or any combination thereof.In certain embodiments, the number of amino acids in the α helix regionof the invention is at least 10 amino acids. In other embodiments, thenumber of amino acids in the α helix region of the invention is 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In other embodiments, thenumber of amino acids in the α helix region of the invention is 24 ormore.

In one embodiment, an α helix region of the invention can be representedby (α helix region type II) R1-Xaa8 Xaa9 Xaa10 R Xaa12 Xaa13 Xaa14 Xaa15Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 P Xaa24 TNT-R1 (SEQ ID NO:30)wherein

Xaa8 is Ala or Val;

Xaa9 is Thr, Met or Leu;

Xaa10 is Gln, Gly, His;

Xaa12 is Leu, or Thr;

Xaa13 is Ala, Thr, Asn, Phe, Tyr, Ser, or Thr;

Xaa14 is Asn, Arg, Ala, Asp, Glu, Gln, Thr, or Gly;

Xaa15 is Phe, Leu, Ser, Glu, Ala, Asp, or Tyr;

Xaa16 is Leu or Asp;

Xaa17 is Val, His, Ser, Phe, or Aib;

Xaa18 is His, Arg, Lys, Orn, hArg, Cit, hLys, Lys(for), or Lys(PEG5000);

Xaa19 is Leu, Ser or Phe;

Xaa20 is Gln or His;

Xaa21 is Thr or Asn;

Xaa22 is Tyr, Val, Phe, Leu or Met;

Xaa24 is Arg or Pro; and

R1 is absent or comprises 1-4 additional amino acids.

Again, it should be remembered that each member in the Markush group, ora combination thereof, is another embodiment of the invention and is notto be read as a single unit. It is further contemplated that the α helixregion may contain no more than one, two, three, four, five, six, seven,eight, nine, or ten modifications such as substitutions, insertions,deletions, and/or derivatizations of the compounds described herein. Forexample, in certain embodiments, the compounds of α helix region type IImay have deletions at the C-terminal end resulting in the deletion ofposition 27, 26, 25, 24, or 22. In other embodiments, however, thedeletions do not remove amino acids of positions 19, 20, 21, or 22.

Examples of an α helix region of type II include, but is not limited to(8Val 9Leu 10Gly) 11-15 hAmylin 16-27 sCT, (8Val 9Leu 10Gly) 11-15hAmylin (18Arg) 16-27 sCT, 8-12 hAmylin (18Arg) 13-27 sCT, 8-18 hAmylin19-23 sCT, 8-18 HAmylin 19-27 sCT, (15Glu 18Arg) 8-18 hAmylin 19-24 sCT,(14Arg 15Ser) 8-18 hAmylin 19-22 sCT, (13Ala 14Ala 15Ala) 8-18 hAmylin19-27 sCT, (13Ala 14Asp 15Ala) 8-18 hAmylin 19-22 sCT, (13Ala 14Asp)8-18 hAmylin 19-23 sCT, (13Ala 14Asp) 8-18 hAmylin 19-27 sCT, (13Ala14Ala) 8-18 hAmylin 19-22 sCT, (13Ala 14Glu) 8-18 hAmylin 19-22 sCT,(13Thr 14Asp 15Tyr) 8-18 hAmylin 19-22 sCT, (13Ala 14Gln) 8-18 hAmylin19-22 sCT, (13Asn 14Glu 15Tyr) 8-18 hAmylin 19-27 sCT, (13Phe 14Asp)8-18 hAmylin 19-27 sCT, (13Ala 14Asp) 8-18 hAmylin (15Glu 18Arg) 8-18hAmylin 19-24 sCT, (19Phe 22Phe) 19-27 sCT, (13Ala 14Asp) 8-18 hAmylin(19Phe 20His 22Phe) 19-27 sCT, (13Ala 14Asp) 8-18 hAmylin (19Phe 22Phe)19-27 sCT, (9Thr 10His) 8-18 hAmylin 19-22 sCT, (9Thr 10His 14Gly 15Leu17Ser 18Arg) 8-19 hAmylin 20-23 sCT, 8-18 hAmylin (21Asn 22Phe 23Val)19-23 sCT, 8-18 hAmylin (22Met) 19-27 sCT, 8-18 hAmylin (22Val) 19-27sCT, (9Met 12Thr 13Tyr 14Thr 15Glu 16Asp 17Phe) 8-17 hAmylin (18Arg)18-20 sCT). In other embodiments, novel compounds include variations ofthe above exemplary compounds with the α helix terminating atcorresponding to 22, 23, 24, 25, 26 or 27 of sCT. In other words,compound 8-18 hAmylin 19-24 sCT is also specifically described as thiscompound is merely 8-18 hAmylin 19-27 sCT described above truncated toposition 24. As another example, compound (13Ala 14Asp 15Ala) 8-18hAmylin 19-23 is specifically described because of the above languageapplied to (13Ala 14Asp 15Ala) 8-18 hAmylin 19-22.

In certain embodiments, the C-terminal tail of the invention comprisesamino acids from position 27, 28, 29, 30, 31, 32, or 33 to position 36or 37 of hAmylin. In other embodiments, the C-terminal tail comprisesamino acids from position 27 or 28 to position 32 of sCT; however, whenthe loop region is from a calcitonin or calcitonin analog and the αhelix region is from a calcitonin or calcitonin analog, the lastposition of the C-terminal tail is not Pro, Hyp, homoSerine (Hse) orderivatives of Hse. Alternatively or additionally, the above described αhelix of amylin and calcitonin may further comprise the substitutions ofone or more of (27Tyr) hAmylin, (29Arg) hAmylin, (32Val) hAmylin,(32Thr) hAmylin, (34Glu) hAmylin, (35Lys) hAmylin, (36Phe) hAmylin,(36Ala) hAmylin, (37Phe) hAmylin, (30Asn)_(s)CT, (32Tyr)_(s)CT, or anycombination thereof.

In one embodiment, a C-terminal tail of the invention can be representedby Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 Xaa33 G Xaa35 Xaa36 Xaa37 Xaa38 (SEQ IDNO:31), wherein

Xaa28 is Lys, Tyr, or absent;

Xaa29 is Ser, Pro, or absent;

Xaa30 is Ser, Pro, Arg, or absent;

Xaa31 is Thr, or absent;

Xaa32 is Asn or absent;

Xaa33 is Val, Thr, or absent;

Xaa35 is Ser, Glu

Xaa36 is Asn, Lys, or Gly;

Xaa37 is Thr, Phe, or Ala;

Xaa38 is Tyr, Phe, Pro, or absent;

with the proviso that when the loop region is from a calcitonin orcalcitonin analog and the α helix region is from a calcitonin orcalcitonin analog, the last position of the C-terminal tail is not Pro,Hyp, homoSerine (Hse) or derivatives of Hse.

Again, it should be remembered that each member of the Markush group, ora combination thereof, is another embodiment of the invention and is notto be read as a single unit. It is further contemplated that theC-terminal tail may contain no more than one, two, or threemodifications such as substitutions, insertions, deletions, and/orderivatizations of the compounds described in the previous paragraphs.

Examples of the C-terminal tail of the invention include, but is notlimited to, 27-37 rAmylin, (27Tyr 29Arg 32Thr) 27-37 rAmylin, (29Arg32Thr) 28-37 rAmylin, 30-37 hAmylin, (32Thr) 30-37 hAmylin, (35Lys 36Ala37Phe) 30-37 hAmylin, 30-36 hAmylin, (32Val) 30-36 hAmylin, (34Glu36Phe) 30-36 hAmylin, 31-37 hAmylin, 31-36 hAmylin, 33-36 hAmylin, 33-7hAmylin, 28-32 sCT, (30Asn 32Tyr) 28-32 sCT, and 27-32 sCT. In otherembodiments, the C-terminal tail comprises the amino acid sequenceKSNFVPTN (SEQ ID NO:32) or SNFVPTNV (SEQ ID NO:33).

It is further contemplated that no more than one, two, or threemodifications such as substitutions, insertions, deletions, and/orderivatizations may be made to the C-terminal tail of the invention asdescribed in the preceding paragraphs. The C-terminal tail of the novelcompounds may further comprise modifications or additional amino acidsat the C-terminal end. Such modifications include the addition ofcompounds such as Lys, up to 4 Lys, L-Octylglycine, 4ABU (4-Aminobutyricacid), 9Anc (9-Amiononanoic acid), and/or groups for solubility,stability, or delivery. Examples include 33-37 hAmylin L-octylglycine,33-37 hAmylin 4ABU, and 33-37 hAmylin 9Anc.

In a general aspect, compounds of the invention comprise

(a) any of the loop region of the invention;

(b) any α helix region of the invention; and

(c) any C-terminal tail of the invention, with the proviso that when theloop region is from a calcitonin or calcitonin analog and the α helixregion is from a calcitonin or calcitonin analog, the last position ofthe C-terminal tail is not Pro, Hyp, homoSerine (Hse) or derivatives ofHse.

In another general aspect, compounds of the invention comprise

(a) a loop region comprising Xaa1 or Xaa1 with modifications at theN-terminal end;

(b) an α helix region comprising the α helix region type I or type II;

(c) a C-terminal tail represented by SEQ ID NO:31, with the proviso thatwhen the loop region is from a calcitonin or calcitonin analog and the αhelix region is from a calcitonin or calcitonin analog, the lastposition of the C-terminal tail is not Pro, Hyp, homoSerine (Hse) orderivatives of Hse. The C-terminal end may comprise furthermodifications.

In yet another aspect, compounds of the invention comprise an amino acidsequence of formula I Xaa1 X Xaa3 Xaa4 Xaa5 Xaa6 Y Xaa8 Xaa9 Xaa10 Xaa11Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 (SEQ ID NO:34)wherein

Xaa1 is A, C, hC, D, E, F, I, L, K, hK, R, hR, S, Hse(homoSER), T, G, Q,N, M, Y, W, P,

Hyp (hydroxyProline), H, V or absent;Xaa3 is A, D, E, N, Q, G, V, R, K, hK, hR, H, I, L, M, or absent;Xaa4 is A, I, L, S, Hse, T, V, M, or absent;

Xaa5 is A, S, T, Hse, Y, V, I, L, or M; Xaa6 is T, A, S, Hse, Y, V, I,L, or M; Xaa8 is A, V, I, L, F, or M; Xaa9 is L, T, S, Hse, V, I, or M;Xaa10 is G, H, Q, K, R, N, hK, or hR; Xaa11 is K, R, Q, N, hK, hR, or H;Xaa12 is L, I, V, F, M, W, or Y; Xaa13 is A, F, Y, N, Q, S, Hse, or T;Xaa14 is A, D, E, G, N, K, Q, R, H, hR, or hK; Xaa15 is A, D, E, F, L,S, Y, I, V, or M; Xaa16 is L, F, M, V, Y, or I; Xaa17 is H, Q, N, S,Hse, T, or V; Xaa18 is K, hK, R, hR, H, u (Cit), or n (Orn);

Xaa19 is F, L, S, Hse, V, I, T, or absent;Xaa20 is H, R, K, hR, hK, N, Q, or absent;Xaa21 is T, S, Hse, V, I, L, Q, N, or absent;

Xaa22 is F, L, M, V, Y, or I; Xaa23 is P or Hyp; Xaa24 is P, Hyp, R, K,hR, hK, or H; Xaa25 is T, S, Hse, V, I, L, F, or Y; Xaa26 is N, Q, D, orE; Xaa27 is T, V, S, F, I, or L; Xaa28 is G or A; Xaa29 is S, Hse, T, V,I, L, or Y; Xaa30 is E, G, K, N, D, R, hR, hK, H, or Q; Xaa31 is A, T,S, Hse, V, I, L, F, or Y; and Xaa32 is F, P, Y, Hse, S, T, or Hyp;

wherein X and Y are capable of creating a bond and are independentlyselected residues having side chains which are chemically bonded to eachother to form an intramolecular linkage such as disulfide bonds; amidebond; alkyl acids and alkyl amines which may form cyclic lactams; alkylaldehydes or alkyl halides and alkylamines which may condensed and bereduced to form an alkyl amine or imine bridge; or side chains which maybe connected to form an alkyl, alkenyl, alkynyl, ether or thioetherbond. Alkyl chains may include lower alkyl groups having from about 1 toabout 6 carbon atoms. In certain embodiments, the intramolecular linkagemay be a disulfide, amide, imine, amine, alkyl and alkene bond. Incertain embodiments, X and Y are independently selected from Ser, Asp,Glu, Lys, Orn, or Cys. In certain embodiments, X and Y are Cys and Cys.In other embodiments, X and Y are Ser and Ser. In still otherembodiments, X and Y are Asp and Lys or Lys and Asp.

In yet another aspect, compounds of the invention comprise an amino acidsequence of formula II: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 (SEQID NO:35) wherein

Xaa1 is A, C, D, F, I, K, S, T, or absent;Xaa2 is C, D, S, or absent;Xaa3 is A, D, N, or absent;Xaa4 is A, L, T, or absent;

Xaa5 is A or S; Xaa6 is T, A, S, or V; Xaa7 is C, K, or A; Xaa8 is A, V,L, or M; Xaa9 is L or T; Xaa10 is G, H, or Q;

Xaa11 is K, R, Q, or hArg;

Xaa12 is L, W, or Y; Xaa13 is A, F, N, Q, S, or T; Xaa14 is A, D, E, G,N, K, Q, or R; Xaa15 is A, D, E, F, L, S, or Y; Xaa16 is L, or F; Xaa17is H, Q, S, or V;

Xaa18 is K, R, hArg, u (Cit), or n (Orn);Xaa19 is F, L, S, or absent;Xaa20 is H, Q, or absent;Xaa21 is T, N, or absent;

Xaa22 is F, L, M, V, or Y; Xaa23 is P; Xaa24 is P or R; Xaa25 is T;Xaa26 is N; Xaa27 is T or V; Xaa28 is G; Xaa29 is S; Xaa30 is E, G, K,or N; Xaa31 is A or T; and Xaa32 is F, P, or Y.

In yet another aspect, compounds of the invention comprise an amino acidsequence of formula III: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32, (SEQID NO:36) wherein

Xaa1 is A, C, F, I, K, S, or absent;

Xaa2 is C, D, or S; Xaa3 is A, D or N; Xaa4 is A, L or T; Xaa5 is A orS; Xaa6 is T; Xaa7 is C or K; Xaa8 is A or V; Xaa9 is L or T; Xaa10 isG, H, or Q;

Xaa11 is K, R, or hArg;

Xaa12 is L; Xaa13 is A, F, N, S, or T; Xaa14 is A, D, E, G, N, Q, or R;Xaa15 is A, E, F, L, S, or Y; Xaa16 is L; Xaa17 is H, S, or V;

Xaa18 is K, R, hArg, u (Cit), or n (Orn);

Xaa19 is F, L, or S; Xaa20 is H or Q; Xaa21 is T or N; Xaa22 is F, L, M,V, or Y; Xaa23 is P; Xaa24 is P or R; Xaa25 is T; Xaa26 is N; Xaa27 isT, or V; Xaa28 is G; Xaa29 is S; Xaa30 is E, G, K, or N; Xaa31 is A, orT; and Xaa32 is F, P, or Y.

In a general aspect, the sequence of formula I, II, or III furthercomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more modificationsof substitutions, insertions, deletions, elongations and/orderivatizations. In certain embodiments, the sequence of formula I, II,or III comprises a Val is inserted between amino acids at positions 22and 23. In other embodiments, the sequence of formula I, II, or IIcomprises a Gln is inserted between positions 22 and 23. In still otherembodiments, the sequence of formula I, II, or III comprises a sequenceof Gln-Thr-Tyr (SEQ ID NO:37) between positions 22 and 23. In yet otherembodiments, the sequence of formula I, II, or III comprises a sequenceof Leu-Gln-Thr-Tyr (SEQ ID NO:38) between positions 22 and 23. Inanother general aspect, the modifications of formula I, II, or III maybe at the N-terminal end. In certain embodiments, the N-terminal portionof formula I, II, or III has an added octylglycine. In otherembodiments, the N-terminal portion of formula I, II or III has an addedisocap.

In yet another aspect, compounds of the invention comprise an amino acidsequence of formula IV: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9Xaa10 Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Xaa30 Xaa31 Xaa32 (SEQID NO:39) wherein

Xaa1 is A, C, D, F, K, T, or absent;Xaa2 is A, C, D, S, or absent;Xaa3 is A, D, N, or absent;Xaa4 is A, L, T, or absent;

Xaa5 is A or S; Xaa6 is A, S, T, or V; Xaa7 is A, C, or K; Xaa8 is A, L,M, or V; Xaa9 is L or T; Xaa10 is G, H, or Q; Xaa11 is K, Q, or R; Xaa12is L, W, or Y; Xaa13 is A, N, Q, S, or T; Xaa14 is A, D, E, G, K, N, Q,or R; Xaa15 is A, D, E, F, L, S, or Y; Xaa16 is F or L; Xaa17 is H, Q, Sor V; Xaa18 is K, or R;

Xaa19 is F, L, S, or absent;Xaa20 is H, K, Q, or absent;Xaa21 is Q, T, or absent;

Xaa22 is F, L, or Y; Xaa23 is P; Xaa24 is P or R; Xaa25 is T; Xaa26 isN; Xaa27 is T or V; Xaa28 is G; Xaa29 is S; Xaa30 is E, K or N; Xaa31 isA or T;

Xaa32 is F, Y, or absent;

In a general aspect, the sequence of formula IV further comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more modifications of substitutions,insertions, deletions, elongations and/or derivatizations. In certainembodiments, the sequence of formula I, II, III, or IV comprises adeletion at position 24.

In yet another aspect, compounds of the invention comprise an amino acidsequence comprising

a) a loop region comprising Xaa1;b) an α helix loop type I; andc) a C-terminal tail;

wherein X1 comprises an amino sequence of X Xaa2 Xaa3 Xaa4 Xaa5 Xaa6Xaa7 Y (SEQ ID NO:138) wherein,

Xaa2 is any amino acid or absent;

Xaa3 is Ala, Gly, Ser, Asp or absent;

Xaa4 is Asn, Ala, Asp, Gly or absent;

Xaa5 is Ala, Leu, Thr, or Ser;

Xaa6 is Ala, Ser, or Thr; and

Xaa7 is Ala, Ser, Val, Hse, (S)-2-amio-3-hydroxy-methylbutanoic acid(Ahb), (2S,3R)-2-amino-3hydroxy-methylpentanoic acid (Ahp), D-Thr, Thr,or a derivative thereof;

X and Y are amino acids capable of creating a bond and are independentlyselected residues having side chains which are chemically bonded to eachother to form an intramolecular linkage such as disulfide bonds; amidebond; alkyl acids and alkyl amines which may form cyclic lactams; alkylaldehydes or alkyl halides and alkylamines which may condensed and bereduced to form an alkyl amine or imine bridge; or side chains which maybe connected to form an alkyl, alkenyl, alkynyl, ether or thioetherbond;

the α helical region type I comprises the sequence R1-V L Xaa10 Xaa11 LS Q Xaa15 L Xaa17 Xaa18 L Q T Xaa22 P Xaa24 T N T-R1 (SEQ ID NO:29),wherein

Xaa10 is Gly or Aib;

Xaa11 is Lys, Arg, Orn, hArg, Cit, hLys, or Lys(for);

Xaa15 is Glu or Phe;

Xaa17 is His or Aib;

Xaa18 is Lys, Arg, Orn, hArg, Cit, hLys, Lys(for), Lys(PEG 5000);

Xaa22 is Tyr or Leu;

Xaa24 is Arg or Pro; or

R1 is absent or comprises 1-4 additional amino acids; and

the C-terminal tail comprises the sequence Xaa28 Xaa29 Xaa30 Xaa31 Xaa32Xaa33 G Xaa35 Xaa36 Xaa37 Xaa38 (SEQ ID NO:31), wherein

Xaa28 is Lys, Tyr, or absent;

Xaa29 is Ser, Pro, or absent;

Xaa30 is Ser, Pro, Arg, or absent;

Xaa31 is Thr, or absent;

Xaa32 is Asn or absent;

Xaa33 is Val, Thr, or absent;

Xaa35 is Ser, Glu

Xaa36 is Asn, Lys, or Gly;

Xaa37 is Thr, Phe, or Ala;

Xaa38 is Tyr, Phe, Pro, or absent;

with the proviso that when the loop region is from a calcitonin orcalcitonin analog and the α helix region is from a calcitonin orcalcitonin analog, the last position of the C-terminal tail is not Pro,Hyp, homoSerine (Hse) or derivatives of Hse.

In yet another aspect, compounds of the invention comprise an amino acidsequence comprising

a) a loop region comprising Xaa1;b) an α helix loop type II; andc) a C-terminal tail;

wherein loop region Xaa1 comprises an amino sequence of X Xaa2 Xaa3 Xaa4Xaa5 Xaa6 Xaa7 Y wherein,

-   -   Xaa2 is any amino acid or absent;    -   Xaa3 is Ala, Gly, Ser, Asp or absent;    -   Xaa4 is Asn, Ala, Asp, Gly or absent;    -   Xaa5 is Ala, Leu, Thr, or Ser;    -   Xaa6 is Ala, Ser, or Thr; and    -   Xaa7 is Ala, Ser, Val, Hse, (S)-2-amio-3-hydroxy-methylbutanoic        acid (Ahb), (2S,3R)-2-amino-3hydroxy-methylpentanoic acid (Ahp),        D-Thr, Thr, or a derivative thereof;    -   X and Y are amino acids capable of creating a bond and are        independently selected residues having side chains which are        chemically bonded to each other to form an intramolecular        linkage such as disulfide bonds; amide bond; alkyl acids and        alkyl amines which may form cyclic lactams; alkyl aldehydes or        alkyl halides and alkylamines which may condensed and be reduced        to form an alkyl amine or imine bridge; or side chains which may        be connected to form an alkyl, alkenyl, alkynyl, ether or        thioether bond;

the α helical region type II comprises the sequence R1-Xaa8 Xaa9 Xaa10 RXaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 PXaa24 TNT-R1 (SEQ ID NO:30) wherein

Xaa8 is Ala or Val;

Xaa9 is Thr, Met or Leu;

Xaa10 is Gln, Gly, His;

Xaa12 is Leu, or Thr;

Xaa13 is Ala, Thr, Asn, Phe, Tyr, Ser, or Thr;

Xaa14 is Asn, Arg, Ala, Asp, Glu, Gln, Thr, or Gly;

Xaa15 is Phe, Leu, Ser, Glu, Ala, Asp, or Tyr;

Xaa16 is Leu or Asp;

Xaa17 is Val, His, Ser, Phe, or Aib;

Xaa18 is His, Arg, Lys, Orn, hArg, Cit, hLys, Lys(for), or Lys(PEG5000);

Xaa19 is Leu, Ser or Phe;

Xaa20 is Gln or His;

Xaa21 is Thr or Asn;

Xaa22 is Tyr, Val, Phe, Leu or Met;

Xaa24 is Arg or Pro; and

R1 is absent or comprises 1-4 additional amino acids; and

the C-terminal tail comprises the sequence Xaa28 Xaa29 Xaa30 Xaa31 Xaa32Xaa33 G Xaa35 Xaa36 Xaa37 Xaa38 (SEQ ID NO:31), wherein

Xaa28 is Lys, Tyr, or absent;

Xaa29 is Ser, Pro, or absent;

Xaa30 is Ser, Pro, Arg, or absent;

Xaa31 is Thr, or absent;

Xaa32 is Asn or absent;

Xaa33 is Val, Thr, or absent;

Xaa35 is Ser, Glu

Xaa36 is Asn, Lys, or Gly;

Xaa37 is Thr, Phe, or Ala;

Xaa38 is Tyr, Phe, Pro, or absent.

In still another aspect, compounds of the invention include:

SEQ ID NO: 40 KCNTATCVLGKLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 41KCNTATCVLGRLSQELHRLQTLPRTNTGSNTY SEQ ID NO: 42KCNTATCVLGRLSQELHRLQTYPPTNTGSNTY SEQ ID NO: 43KCNTATCVLGRLSQELHRLQTYPRTNVGSNTY SEQ ID NO: 44KCNTATCVLGRLSQELHRLQTLPPTNVGSNTY SEQ ID NO: 45KCNTATCVLGRLANFLHRLQTYPRTNTGSNTY SEQ ID NO: 46ACNTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 47KCNAATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 48KCNTAACVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 49CANLSTCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 50isocaproyl-STAVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 51CSNASTCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 52CSNLATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 53CSNLSACVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 54KCNTATCVLGRLSQELHKLQTYPRTNTGSNTY SEQ ID NO: 55KCNTATCVLGRLSQELHRLQTYPRTNTGSGTP SEQ ID NO: 56CSALSTCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 57Ac-(Agy)SNLST(Agy)VLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 58Ac-K(Agy)NTAT(Agy)VLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 59Isocaproyl-STAVL(Aib)RLSQELRLQTYPRTNTGSGTP SEQ ID NO: 60Isocaproyl-STAVLG[K(For)]LSQELH[K(For)]LQTYPRTNTGSGTP SEQ ID NO: 61Isocaproyl-STAVL(Aib)[K(For)]LSQEL(Aib)[K(For)]LQTYPRTNTGSNTYSEQ ID NO: 62Isocaproyl-STAVL(Aib)[K(For)]LSQEL(Aib)[K(For)]LQTYPRTNVGSNTYSEQ ID NO: 63 KCNTATCLLQQLQKLLQKLKQYPRTNTGSNTY SEQ ID NO: 64KCNTASCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 65KCNTAVCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 66KCNTATCVLGRLSQELHRYPRTNTGSNTY SEQ ID NO: 67KCNTATCVLGK(For)LSQELHK(For)LQTYPRTNTGSNTY SEQ ID NO: 68KCNTA(d-Thr)CVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 69KCNTA(dAh)CVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 70Ac-ACNTATCVLGRLSQELHK(PEG5000)LQTYPRTNTGSNTY SEQ ID NO: 71KCNTATCVLGRLSQELHRLQTLQTYPRTNTGSNTY SEQ ID NO: 72KCNTATCVLGRLSQELHRLQTLLQTYPRTNTGSNTY SEQ ID NO: 73KCNTATCVLGKLSQELHKLQTYPRTNTGSNTY SEQ ID NO: 74KCNTSTCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 75KCNTATCATQRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 76KCNTATCATQRLSQELHRLQTYPRTNVGSNTY SEQ ID NO: 77KCNTSTCATQRLANELVRLQTYPRTNVGSNTY SEQ ID NO: 78KCNTA(Hse)CVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 79KCNTA(Ahb)CVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 80KCNTA(Ahp)CVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 81KCNTAT(OPO3H2)CVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 82KCNTATCVLG(Orn)LSQELH(Orn)LQTYPRTNTGSNTY SEQ ID NO: 83KCNTATCVLG(Cit)LSQELH(Cit)LQTYPRTNTGSNTY SEQ ID NO: 84KCNTATCVLG(homoK)LSQELH(homoK)LQTYPRTNTGSNTY SEQ ID NO: 85L-OctylglycineKCNTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 86N-3,6-dioxaoctanoyl-CNTATCVLGRLSQELHRLQTVPRTNTGSNTY SEQ ID NO: 87KCNTATCMLGRYTQDFHRLQTYPRTNTGSNTY SEQ ID NO: 88DSNLSTKVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 89KDNTATKVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 90CNTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 91KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY(9Anc) SEQ ID NO: 92KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY(L-octylglycine) SEQ ID NO: 93N-isocaproyl-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 94KCNTATCVLG(homoR)LSQELH(homoR)LQTYPRTNTGSNTY SEQ ID NO: 95FCNTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 96KCNTATCVLGRLSQELH(Cit)LQTYPRTNTGSNTY SEQ ID NO: 97KCNTATCVLGRLSQELH(Orn)LQTYPRTNTGSNTY SEQ ID NO: 98ICNTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 991-Octylglycine-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 100Isocaproyl-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 101KCNTATCVLG(Cit)LSQELHRLQTYPRTNTGSNTY SEQ ID NO: 102KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY(4ABU) SEQ ID NO: 103Isocaproyl-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY(4ABU) SEQ ID NO: 104KCNTSTCATQRLANELVRLQTYPRTNVGSEAF SEQ ID NO: 105KCNTATCVLGRLSQELHRLQTYPTNVGSEAF SEQ ID NO: 106KCNTATCVLGRLSRSLHRLQTYPRTNTGSNTY SEQ ID NO: 107KCNTATCVTHRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 108KCNTATCVLGRLADFLHRLQTYPRTNTGSNTY SEQ ID NO: 109CNTATCVLGRLSQELHRLQTYPRTNTGSNT SEQ ID NO: 110KCNTATCVLGRLSQELHRLQNFVPRTNTGSNTY SEQ ID NO: 111KCNTATCVLGRLSQELHRLQTYPRTNTGSETF SEQ ID NO: 112ACDTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 113KCNTATCVLGRLSQELHRLQTYPRTNTGSKAF SEQ ID NO: 114KCDTATCVTHRLAGLLSRSQTYPRTNTGSNTY SEQ ID NO: 115KCNTATCVLGRLADALHRLQTYPRTNTGSNTY SEQ ID NO: 116KCNTATCVLGRLAAFLHRLQTYPRTNTGSNTY SEQ ID NO: 117SCNTATCVLGRLADFLHRLQTYPRTNTGSNTY SEQ ID NO: 118KCNTATCVLGRLSQELHRLQTMPRTNTGSNTY SEQ ID NO: 119KCNTATCVLGRLSQELHRLQTVPRTNTGSNTY SEQ ID NO: 120KCNTATCVLGRLNEYLHRLQTYPRTNTGSNTY SEQ ID NO: 121SCNTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 122KCNTATCVLGRLTEFLHRLQTYPRTNTGSNTY SEQ ID NO: 123KCNTATCVLGRLAEFLHRLQTYPRTNTGSNTY SEQ ID NO: 124KCNTATCVLGRLTDYLHRLQTYPRTNTGSNTY SEQ ID NO: 125KCNTATCVLGRLAQFLHRLQTYPRTNTGSNTY SEQ ID NO: 126KCNTATCVLGRLADFLHRFQTFPRTNTGSNTY SEQ ID NO: 127KCNTATCVLGRLADFLHRFHTFPRTNTGSNTY SEQ ID NO: 128KCNTATCVLGRLADFLHRFQTFPRTNTGSGTP SEQ ID NO: 129CNTATCVLGRLADFLHRLQTYPRTNTGSNTY SEQ ID NO: 130KCDTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 131KCNTATCVLGRLFDFLHRLQTYPRTNTGSNTY SEQ ID NO: 132KCNTATCVLGRLAAALHRLQTYPRTNTGSNTY SEQ ID NO: 133TCDTATCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 134CSNLSTCATQRLANELVRLQTYPRTNVGSNTY SEQ ID NO: 135KCNTATCATQRLANELVRLQTYPRTNVGSNTY SEQ ID NO: 136CSNLSTCVLGRLSQELHRLQTYPRTNTGSNTY SEQ ID NO: 137KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY

In still another aspect, compounds of the invention include biologicallyactive fragments of SEQ ID NOS:40 to 137. Biologically active fragmentsmay comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or more amino acids. In certain embodiments, the amino acid sequencesof SEQ ID NOs:40 to 137 comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more modifications such as substitutions, insertions, deletions,and/or derivatizations. In other embodiments, the amino acid sequencesof SEQ ID NOs:40 to 137 has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 modifications such as substitutions, insertions, deletions, and/orderivatizations. In still another aspect of the invention, compounds ofthe invention include those having at least 75, 80, 85, 90, 95, or 97%amino acid sequence identity to any of SEQ ID NOS: 40 to 137. Percentidentity is determined by analysis with the AlignX module in Vector NTI(Invitrogen; Carlsbad Calif.). It is intended that each percent identitydescribed, or reference to biologically active fragments ormodifications be applied to each SEQ ID NO: individually. For example,each embodiment described, fragments, modification, or % identity isapplicable to SEQ ID NO:40, 41, 42, 43, 44, etc., or to any group of SEQID NOs:. Moreover, the compounds of the invention include the compounds1-127 described in Example 3.

In another general aspect, compounds of the invention may act as anagonist for at least one biological effect of calcitonin, amylin, and/orCGRP herein disclosed or bind to at least one of the receptors ofamylin, calcitonin, or CGRP.

In still another general aspect, the compounds of the invention may beuseful for reducing food intake, reducing appetite, inducing satiety,reducing nutrient availability, causing weight loss, affecting bodycomposition, altering body energy content or energy expenditure,improving lipid profile (including reducing LDL cholesterol andtriglyceride levels and/or changing HDL cholesterol levels), slowinggastrointestinal motility, delay gastric emptying, moderating thepostprandial blood glucose excursions, preventing or inhibiting glucagonsecretion, and decreasing blood pressure.

Thus, in certain embodiments, the methods of the invention are usefulfor treating or preventing conditions or disorders which can bealleviated by reducing nutrient availability comprising administering tosaid subject a therapeutically or prophylactically effective amount of acompound of the invention. Such conditions and disorders include, butare not limited to, eating disorders, insulin-resistance, obesity,abnormal postprandial hyperglycemia, diabetes of any kind, includingType I, Type II, and gestational diabetes, Metabolic Syndrome, DumpingSyndrome, hypertension, dyslipidemia, cardiovascular disease,hyperlipidemia, sleep apnea, cancer, pulmonary hypertension,cholecystitis, and osteoarthritis.

Non-limiting examples of a cardiovascular condition or disease arehypertension, myocardial ischemia, and myocardial reperfusion. Compoundsof the invention may also be useful in treating or preventing otherconditions associated with obesity including stroke, cancer (e.g.,endometrial, breast, prostate, and colon cancer), gallbladder disease,sleep apnea, reduced fertility, and osteoarthritis, (see Lyznicki et al,Am. Fam. Phys. 63:2185, 2001). In other embodiments, compounds of theinvention may be used to alter body composition for aesthetic reasons,to enhance one's physical capabilities, or to produce a leaner meatsource.

In another general aspect, compounds of the invention may be used toinhibit the secretion of ghrelin. Accordingly, compounds of theinvention may be utilize this mechanism to treat or prevent ghrelinrelated disorders such as Prader-Willi syndrome, diabetes of all typesand its complications, obesity, hyperphagia, hyperlipidemia, or otherdisorders associated with hypernutrition.

In another general aspect, it is now recognized that amylin and amylinagonists, including compounds of the invention, may be useful fortreating or preventing Barrett's esophagus, Gastroesophageal RefluxDisease (GERD) and conditions associated therewith. Such conditions caninclude, but are not limited to, heartburn, heartburn accompanied byregurgitation of gastric/intestinal contents into the mouth or thelungs, difficulty in swallowing, coughing, intermittent wheezing andvocal cord inflammation (conditions associated with GERD), esophagealerosion, esophageal ulcer, esophageal stricture, Barrett's metaplasia(replacement of normal esophageal epithelium with abnormal epithelium),Barrett's adenocarcinoma, and pulmonary aspiration. Amylin and amylinagonists, including compounds of the invention, have anti-secretoryproperties, such as inhibition of gastric acids, inhibition of bileacids, and inhibition of pancreatic enzymes. Moreover, amylin has beenfound to have a gastroprotective effect. Accordingly, these propertiesof amylin, amylin agonists and compounds of the invention may renderthem particularly useful in the treatment or prevention of Barrett'sesophagus, and/or GERD and related or associated conditions as describedherein.

In another general aspect, compounds of the invention may further beuseful for treating or preventing pancreatitis, pancreatic carcinoma,and gastritis. Moreover, compounds of the invention may be useful in thetreatment and prevention of pancreatitis in patients who have undergoneendoscopic retrograde cholangiopancreatography (ERCP). It has furtherbeen discovered that amylin and amylin agonists, including compounds ofthe invention, may have a surprisingly superior therapeutic effect whencombined with somatostatin. Accordingly, in certain embodiments, methodsfor treating or preventing pancreatitis comprise administering amylin,and amylin agonists, including compounds of the invention, andadministering somatostatin and somatostatin agonists to a subject. Inother embodiments, methods for treating or preventing pancreatitiscomprise administering compounds of the invention and administeringsomatostatin and somatostatin agonists.

In another general aspect, compounds of the invention may also be usefulfor decreasing bone resorption, decreasing plasma calcium, and inducingan analgesic effect. Accordingly, compounds of the invention may beuseful to treat bone disorder such as osteopenia and osteoporosis. Inyet other embodiments, compounds of the invention may be useful to treatpain and painful neuropathy.

In still another general aspect, the compounds of the invention may beused as part of a combination therapy. In certain embodiments, compoundsof the invention may be used with other commercially available diet aidsor other anti-obesity agents, such as, by way of example, PYY and PYYagonists, GLP-1 and GLP-1 agonists, a DPPIV inhibitor, CCK and CCKagonists, exendin and exendin agonists, and leptin and leptin agonists.In other embodiments, compounds of the invention may be used with otheranalgesics, immune suppressors, or other anti-inflammatory agents.

In still another general aspect, novel pharmaceutical compositionscomprising the compounds of the invention are described as well asmethods for using them. In certain embodiments, pharmaceuticalcompositions may comprise at least 0.01% to 5% w/v. In certainembodiments, the pH of the composition can be from about 3.0 to about6.0. In certain embodiments, the buffer may be acetate, phosphate,citrate, or glutamate. In certain embodiments, composition furthercomprises a carbohydrate or polyhydric alcohol tonicifier. In certainembodiments, the composition further comprises a preservative selectedfrom the group consisting of m-cresol, benzyl alcohol, parabens andphenol. In still other embodiments, administration of the compounds in adose in the range of about 0.05 mg/kg to about 2 mg/kg. For example, adaily dose may be 1 μg to about 5 mg per day. In certain embodiments,exemplary modes of delivery can be injection, infusion, absorption(mucosal), and inhalation. Routes of administration can beintramuscular, intravenous, subcutaneous, transdermal, transmucosal,oral, nasal, or by pulmonary inhalation.

Also contemplated as part of the invention are nucleotides that encodethe amino acid sequence herein described, vectors containing thenucleotides, host cells for propagating nucleotides and/or expressingthe polypeptides encoded by the nucleotides, antibodies directed to thenovel compounds, and their uses in screening or detection/diagnosis of acondition such as those described herein in a subject.

These and other aspects of the invention will be more clearly understoodwith reference to the following preferred embodiments and detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates a dose-dependent reduction in food consumption inover night fasted mice to doses of an exemplary compound of theinvention (Food Intake Assay).

FIG. 2A illustrates a decreased caloric intake in fattened (diet-inducedobese, or DIO) rats with continuous peripheral infusion of either avehicle of exemplary compounds of the invention over a period of twoweeks.

FIG. 2B illustrates a reduction in body weight over the correspondingtime period.

FIG. 3 depicts effects on body composition by an exemplary compound ofthe invention.

FIG. 4A depicts effects on gastric emptying by an exemplary compound ofthe invention.

FIG. 4B depicts effect of hypocalcemia by an exemplary compound of theinvention.

FIG. 5 depicts effects on triglycerides by an exemplary compound of theinvention.

FIGS. 6A-6C depict the effect on triglycerides after 1 week (FIG. 6A), 2weeks (FIG. 6B), and 3 weeks (FIG. 6C) by an exemplary compound (SEQ IDNO:137) of the invention.

FIGS. 7A-7E depict the dose effect on triglycerides at baseline (FIG.7A) and after 2 weeks (FIG. 7B), 4 weeks (FIG. 7C), 6 weeks (FIG. 7D),and 8 weeks (FIG. 7E) by an exemplary compound (SEQ ID NO:137) of theinvention.

FIG. 8A depicts the effect on ghrelin by amylin following theadministration of a pentagastrin injection.

FIGS. 8B-8C depict the comparative effect on ghrelin by saline and 30μg/kg of amylin without a pentagastrin injection (FIG. 8B) and followinga pentagastrin injection (FIG. 8C).

FIG. 9A depicts the effect of plasma amylase, a marker on pancreaticfunction, by an exemplary compound (SEQ ID NO:137) of the invention.

FIG. 9B depicts the effect of plasma lipase, a marker on pancreaticfunction, by an exemplary compound (SEQ ID NO:137) of the invention.

FIG. 10A depicts the effect on gastric acid secretion by amylin.

FIG. 10B depicts the dose response effect on gastric acid secretion byamylin.

FIG. 11A depicts the gastric mucosal protection of amylin.

FIG. 11B depicts the gastric mucosal injury score of amylin.

FIG. 11C depicts the dose response curve of amylin.

DETAILED DESCRIPTION

The present invention relates to novel amylin family compounds oragonists (also referred to as novel compounds and compounds of theinvention). These novel compounds may be useful in treating orpreventing conditions such as metabolic disorders, vascular disorders,renal disorders, and/or gastrointestinal disorders. The previous sectionprovides the structure of the compounds. The novel compounds may furtherbe described as having desirable characteristic such as comparable orhigher activity in the treatment and/or prevention of metabolicconditions and disorders and those referenced above, as compared toamylin, calcitonin, and/or CGRP. In other embodiments, the compounds ofthe invention may not have comparable or higher activity but may haveincreased stability or solubility, fewer side effects, combination ofbiological activities, quicker onset of activity, longer duration ofactivity, and/or ease in manufacturing, formulating, or use than amylin,calcitonin or CGRP.

By an amylin activity is meant that a compound demonstrates similarphysiological characteristics as amylin, such as those described in theinstant specification, for example, reducing food intake. The compoundsof the present invention may be capable of binding to or otherwisedirectly or indirectly interacting with an amylin receptor, or otherreceptor or receptors with which amylin itself may interact to elicit abiological response, e.g., reducing food intake.

By a calcitonin activity is meant that a compound demonstrates similarphysiological characteristics as calcitonin, such as those described inthe instant specification, for example, inhibiting osteoclast function.The compounds of the present invention may be capable of binding to orotherwise directly or indirectly interacting with a CT receptor, orother receptor or receptors with which calcitonin itself may interact toelicit a biological response, e.g., inhibiting osteoclast function.

By a CGRP activity is meant that a compound demonstrates similarphysiological characteristics as CGRP, such as those described in theinstant specification, for example, eliciting a vasodilatory effect. Thecompounds of the present invention may be capable of binding to orotherwise directly or indirectly interacting with a CGRP receptor, orother receptor or receptors with which CGRP itself may interact toelicit a biological response, e.g., eliciting a vasodilatory effect.

The compounds of the invention may also include biologically activefragments of the larger peptides described herein that retain activity.Therefore, examples of desirable activities possessed by the compoundsof the invention include (1) having activity in a food intake, gastricemptying, pancreatic secretion, blood pressure, heart rate or weightloss assay similar to amylin, calcitonin, or CGRP, and/or (2) binding ina receptor binding assay for amylin, calcitonin, or CGRP. Some exemplaryassays are provided in Example 1.

Compounds of the invention may further have a particular bindingprofile. For example, it has been reported that the biological actionsof amylin, calcitonin, and CGRP are mediated via binding to two closelyrelated type II G protein-coupled receptors (GPCRs), the calcitoninreceptor (CTR) and the calcitonin receptor like receptor (CRLR). Cloningand functional studies have shown that CGRP and amylin interact withdifferent combinations of CTR or the CRLR and the receptor activitymodifying protein (RAMP). Many cells express multiple RAMPs. It isbelieved that co-expression of RAMPs and either the CTR or CRLR isrequired to generate functional receptors for calcitonin, CGRP, andamylin. The RAMP family comprises three members (RAMP1, -2, and -3),which share less then 30% sequence identity, but have a commontopological organization. Co-expression of CRLR and RAMP1 leads to theformation of a receptor for CGRP as does co-expression of CRLR andRAMP3. Co-expression of hCTR2 and RAMP1 leads to the formation of areceptor for amylin and CGRP. Co-expression of hCTR2 and RAMP3 leads tothe formation of a receptor for amylin.

Accordingly, the compounds of the invention for use in the methods ofthe present invention may demonstrate affinity to receptors of amylin,CGRP, and calcitonin in the amylin family. Compounds of the inventionmay show a significant affinity for binding to the amylin receptor, aswell as the ability to bind to other receptors such as calcitonin andCGRP receptors. Compounds of the invention may bind an amylin receptorwith an affinity of greater than 20 nM, 10 nM, 5 nM, 1 nM, and morepreferably with an affinity of greater than 0.10 nM. In addition,compounds of the present invention may also bind with similar affinitiesto the calcitonin and CGRP receptors, but with a lower affinity at theCGRP receptor. In other embodiments, compounds of the invention may bindto a calcitonin receptor with an affinity of greater than 20 nM, 10 nM,or 1 nM. In still other embodiments, compounds of the invention may bindto a CGRP receptor with an affinity of greater than about 1 μM, 700 nM,or 500 nM. Compounds of the invention may also bind to all threereceptors to varying degrees. Accordingly, it is contemplated thatcompounds of the invention may have a binding profile with a particularbinding affinity for each receptor from the ones described herein.

Compounds of the invention, including those of formula I, II, III, IV,biologically active fragments of SEQ ID NOS: 40 to 137, those having atleast 75, 80, 82 85, 87, 90, 92, 95, or 97% amino acid sequence identityto any of SEQ ID NOS: 40 to 237, and biologically active fragmentsthereof may retain at least about 25%, preferably about 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, or 99% percent of the biological activityof amylin, calcitonin, CGRP or compounds having the sequence of SEQ IDNOS:40 to 137, or Compounds 1-137 in Example 3, with respect to thereduction of food intake or one of the other activities describedherein, e.g., Table 1. In another embodiment, compounds of the inventionexhibit improved biological activity. Preferably, the novel compoundsexhibit at least about 110%, 125%, 130%, 140%, 150%, 200%, or more ofthe biological activity of amylin, calcitonin, CGRP or the compoundshaving the sequence of SEQ ID NOS:40 to 137, or Compounds 1-137 inExample 3, with respect to the reduction of food intake or one of theother activities described herein, e.g, Table 1. For example, adesirable compound of the invention is one having an activity in one ofthe assays described herein (food intake, weight reduction assay,gastric emptying, triglycerides, pancreatitis, ghrelin, or calcium)which is greater than the activity of amylin, calcitonin, or CGRP inthat same assay.

As illustration only, desirable compounds of the invention maydemonstrate an ability to reduce cumulative food intake more than 5%over administration of the vehicle, preferably more than 15%, morepreferably more than 25%, even more preferably more than 35% or 40% mostpreferably more than 50% over the vehicle.

In still another general aspect, the compounds of the invention may beuseful for reducing food intake, reducing appetite, inducing satiety,reducing nutrient availability, causing weight loss, affecting bodycomposition, altering body energy content or energy expenditure,improving lipid profile (including reducing LDL cholesterol andtriglyceride levels and/or changing HDL cholesterol levels), slowinggastrointestinal motility, delay gastric emptying, moderating thepostprandial blood glucose excursions, preventing or inhibiting glucagonsecretion, and decreasing blood pressure. Exemplary assays for effectson food intake, weight reduction, gastric emptying, triglycerides, andbody composition are described in at least Examples 3, 4, 5, 6, and 7.

Thus, in certain embodiments, the methods of the invention are usefulfor treating or preventing conditions or disorders which can bealleviated by reducing nutrient availability comprising administering tosaid subject a therapeutically or prophylactically effective amount of acompound of the invention. Such conditions and disorders include, butare not limited to, eating disorders, insulin-resistance, obesity,abnormal postprandial hyperglycemia, diabetes of any kind, includingType I, Type II, and gestational diabetes, Metabolic Syndrome, DumpingSyndrome, hypertension, dyslipidemia, cardiovascular disease,hyperlipidemia, sleep apnea, cancer, pulmonary hypertension,cholecystitis, and osteoarthritis.

Non-limiting examples of a cardiovascular condition or disease arehypertension, myocardial ischemia, and myocardial reperfusion. Compoundsof the invention may also be useful in treating or preventing otherconditions associated with obesity including stroke, cancer (e.g.,endometrial, breast, prostate, and colon cancer), gallbladder disease,sleep apnea, reduced fertility, and osteoarthritis, (see Lyznicki et al,Am. Fam. Phys. 63:2185, 2001). In other embodiments, compounds of theinvention may be used to alter body composition for aesthetic reasons,to enhance one's physical capabilities, or to produce a leaner meatsource.

In another general aspect, compounds of the invention may be used toinhibit the secretion of ghrelin. Accordingly, compounds of theinvention may be utilize this mechanism to treat or prevent ghrelinrelated disorders such as Prader-Willi syndrome, diabetes of all typesand its complications, obesity, hyperphagia, hyperlipidemia, or otherdisorders associated with hypernutrition. An exemplary assay for effectson ghrelin is described in Example 8.

In another general aspect, it is now recognized that amylin and amylinagonists, including compounds of the invention, may be useful fortreating or preventing Barrett's esophagus, Gastroesophageal RefluxDisease (GERD) and conditions associated therewith. Such conditions caninclude, but are not limited to, heartburn, heartburn accompanied byregurgitation of gastric/intestinal contents into the mouth or thelungs, difficulty in swallowing, coughing, intermittent wheezing andvocal cord inflammation (conditions associated with GERD), esophagealerosion, esophageal ulcer, esophageal stricture, Barrett's metaplasia(replacement of normal esophageal epithelium with abnormal epithelium),Barrett's adenocarcinoma, and pulmonary aspiration. Amylin and amylinagonists, including compounds of the invention, have anti-secretoryproperties, such as inhibition of gastric acids, inhibition of bileacids, and inhibition of pancreatic enzymes. Accordingly, theseproperties of amylin, amylin agonists and compounds of the invention mayrender them particularly useful in the treatment or prevention ofBarrett's esophagus, and/or GERD and related or associated conditions asdescribed herein. Exemplary assays showing effects on gastric acidsecretion and gastroprotective effect are described in Examples 10 and11.

In another general aspect, compounds of the invention may further beuseful for treating or preventing pancreatitis, pancreatic carcinoma,and gastritis. Moreover, compounds of the invention may be useful in thetreatment and prevention of pancreatitis in patients who have undergoneendoscopic retrograde cholangiopancreatography (ERCP). It has furtherbeen discovered that amylin and amylin agonists, including compounds ofthe invention, may have a surprisingly superior therapeutic effect whencombined with somatostatin. Accordingly, in certain embodiments, methodsfor treating or preventing pancreatitis comprise administering amylin,and amylin agonists, including compounds of the invention, andadministering somatostatin and somatostatin agonists to a subject. Inother embodiments, methods for treating or preventing pancreatitiscomprise administering compounds of the invention and administeringsomatostatin and somatostatin agonists. An exemplary assay showingeffects on pancreatic function is described in Example 9.

In another general aspect, compounds of the invention may also be usefulfor decreasing bone resorption, decreasing plasma calcium, and inducingan analgesic effect. Accordingly, compounds of the invention may beuseful to treat bone disorder such as osteopenia and osteoporosis. Inyet other embodiments, compounds of the invention may be useful to treatpain and painful neuropathy. Exemplary assay showing effects on calciumlevels are provided in Example 6.

In the methods of the present invention, the polypeptides may beadministered separately or together with one or more other compounds andcompositions that exhibit a long term or short-term action to reducenutrient availability, including, but not limited to other compounds andcompositions that comprise an amylin or amylin analog agonist, salmoncalcitonin, a cholecystokinin (CCK) or CCK agonist, a leptin (OBprotein) or leptin agonist, an exendin or exendin analog agonist, aGLP-1 or GLP-1 analog agonist, a DPPIV inhibitor, a PYY or PYY analog,AFP-6 (intermedin) or AFP-6 agonist, Urocortin or Urocortin agonist, orAdrenomedullin or Adrenomeullin agonist. Suitable amylin agonistsinclude, for example, [25,28,29 Pro-]-human amylin (also known as“pramlintide,” and described in U.S. Pat. Nos. 5,686,511 and 5,998,367).The CCK used is preferably CCK octopeptide (CCK-8). Leptin is discussedin, for example, (Pelleymounter, Cullen et al., Science 269: 540-543(1995); Halaas, Gajiwala et al., Science 269: 543-6 (1995); Campfield,Smith et al., Science 269: 546-549 (1995)). Suitable exendins includeexendin-3 and exendin-4, and exendin agonist compounds include, forexample, those described in PCT Publications WO 99/07404, WO 99/25727,and WO 99/25728. Suitable PYY polypeptides and analogs include thosedescribed in U.S. Application Nos.: 60/543,406 and 60/543,407.

While “obesity” is generally defined as a body mass index over 30, forpurposes of this disclosure, any subject, including those with a bodymass index of less than 30, who needs or wishes to reduce body weight isincluded in the scope of “obese.”

Preparation of Compounds of the Invention

The compounds of the invention described herein may be prepared usingstandard recombinant techniques or chemical peptide synthesis techniquesknown in the art, e.g., using an automated or semi-automated peptidesynthesizer, or both. Likewise, the derivatives of the polypeptides ofthe invention may be produced using standard chemical, biochemical, orin vivo methodologies.

The compounds of the invention can be synthesized in solution or on asolid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. See, e.g., Stewart and Young, SolidPhase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984); Tam etal., J. Am. Chem. Soc. 105: 6442 (1983); Merrifield, Science 232: 341-7(1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer,eds., Academic Press, New York, 1-284 (1979). Solid phase peptidesynthesis may be carried out with an automatic peptide synthesizer(e.g., Model 430A, Applied Biosystems Inc., Foster City, Calif.) usingthe NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry (see, AppliedBiosystems User's Manual for the ABI 430A Peptide Synthesizer, Version1.3B Jul. 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc.,Foster City, Calif.) with capping. Peptides may also be assembled usingan Advanced ChemTech Synthesizer (Model MPS 350, Louisville, Ky.).Peptides may be purified by RP-HPLC (preparative and analytical) using,e.g., a Waters Delta Prep 3000 system and a C4, C8, or C18 preparativecolumn (10 μL, 2.2×25 cm; Vydac, Hesperia, Calif.). The active proteincan be readily synthesized and then screened in screening assaysdesigned to identify reactive peptides.

Besides the classical step-by-step synthesis, convergent solid phasepeptide synthesis (also known as hybrid approach or as fragmentcondensation method) has been developed for the preparation of complexand difficult peptides and small proteins. According to this method,suitably protected peptide fragments spanning the entire peptidesequence and prepared on the solid phase are condensed, either on solidsupport or in solution, to the target peptide. The availability of newresins and resin handles has opened up the possibility of synthesizingfully protected peptide segments rapidly by the solid-phase technique.The approach is particularly attractive for the manufacture of largemolecules, since it combines the advantages of both the solid-phase andthe solution-phase methods. Production cycle times are short, comparedwith solution-phase methodologies, and yields and purities are oftenhigher. Additionally, the scale-up is a lot easier, and avoids many ofthe aggregation problems often encountered in solid-phase synthesis oflong peptides.

In another embodiment, the synthetic strategy uses convergent fragmentcondensation. Convergent fragment condensation is a superior method ofproducing large, high quality peptides over standard solid or standardliquid phase synthesis. With such methods fragment can be synthesized inparallel cutting down on the time to synthesize as well as ensuringquality. As peptides grow longer, there is more risk of side reactionsand incomplete synthesis. It has been recognized that peptide sequencesof the invention can have ideally located Glycine and Proline whichpromote a fragment approach because those two amino acids are not ableto racemize during fragment coupling and show very efficientcondensation rates. Besides the fact that a straight forward solid phasesynthesis presents major scale-up problems and is therefore probably notsuitable for large scale synthesis, a fragment approach is much morecontrollable and provides the opportunity to purify intermediates. Thedisulfide bridge is located in the first fragment only and can thereforebe formed at the fragment stage with the totally protected precursor.Since this approach presents a lot of advantages in terms of strategyand scale, it will shorten the synthesis time because all the fragmentscan be synthesized simultaneously. This is a great tool to minimize riskand to reduce costs. The following fragments are depicted to show askeletal structure of desirable fragments where the would allow forimplementation of this strategy:

-   Fragment 1: Boc-X (Boc)-X-X (Trt)-X (tBu)-X-X(tBu)-X-X-X-Gly-OH    Cyclic (cyclization would take place prior to fragment coupling)-   Fragment 2:    Fmoc-K(Pbf)-X-X(tBu)-X(Trt)-X(OtBu)-X-X(Trt)-X(Pbf)-X-X(Trt)-X(tBu)-X(tBu)-Pro-OH-   Fragment 3:    Fmoc-X(Pbf)-X(tBu)-X(Trt)-X(tBu)-X-X(tBu)-X(Trt)-Thr(tBu)-OH-   Fragment 4: H-Tyr(tBu)-NH₂    Based on these teaching it would understandable to one of skill in    the art which compounds of the invention would be ideal for such    synthesis. Evolving technology may make it possible to only have    three fragments, i.e., the 4^(th) fragment need not be created    separately from the 3^(rd).

The compounds of the present invention may alternatively be produced byrecombinant techniques well known in the art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor(1989). These polypeptides produced by recombinant technologies may beexpressed from a polynucleotide. These polynucleotide sequences mayincorporate codons facilitating transcription and translation of mRNA inmicrobial hosts. Such manufacturing sequences may readily be constructedaccording to the methods well known in the art. See, e.g., WO 83/04053.The polynucleotides above may also optionally encode an N-terminalmethionyl residue. Non-peptide compounds useful in the present inventionmay be prepared by art-known methods. For example, phosphate-containingamino acids and peptides containing such amino acids may be preparedusing methods known in the art. See, e.g., Bartlett and Landen, Bioorg.Chem. 14: 356-77 (1986).

A variety of expression vector/host systems may be utilized to containand express the coding sequence of the novel compounds. These includebut are not limited to microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transfected with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withbacterial expression vectors (e.g., Ti or pBR322 plasmid); or animalcell systems. Mammalian cells that are useful in recombinant proteinproductions include but are not limited to VERO cells, HeLa cells,Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), WI38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells.Exemplary protocols for the recombinant expression of the protein aredescribed herein below.

As such, polynucleotide sequences provided by the invention are usefulin generating new and useful viral and plasmid DNA vectors, new anduseful transformed and transfected procaryotic and eucaryotic host cells(including bacterial, yeast, and mammalian cells grown in culture), andnew and useful methods for cultured growth of such host cells capable ofexpression of the present polypeptides. The polynucleotide sequencesencoding the novel compounds herein may also be useful for gene therapy.

The present invention also provides for processes for recombinant DNAproduction of the novel compounds. Provided is a process for producingthe polypeptides from a host cell containing nucleic acids encoding thenovel compounds comprising: (a) culturing said host cell containingpolynucleotides encoding such polypeptides under conditions facilitatingthe expression of such DNA molecule; and (b) obtaining the novelcompounds.

Host cells may be prokaryotic or eukaryotic and include bacteria,mammalian cells (such as Chinese Hamster Ovary (CHO) cells, monkeycells, baby hamster kidney cells, cancer cells or other cells), yeastcells, and insect cells.

Mammalian host systems for the expression of the recombinant proteinalso are well known to those of skill in the art. Host cell strains maybe chosen for a particular ability to process the expressed protein orproduce certain post-translation modifications that will be useful inproviding protein activity. Such modifications of the polypeptideinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing, which cleaves a “prepro” form of theprotein, may also be important for correct insertion, folding and/orfunction. Different host cells, such as CHO, HeLa, MDCK, 293, WI38, andthe like, have specific cellular machinery and characteristic mechanismsfor such post-translational activities, and may be chosen to ensure thecorrect modification and processing of the introduced foreign protein.

Alternatively, a yeast system may be employed to generate the novelcompounds of the invention. The coding region of the DNA encoding thenovel compound is amplified by PCR. A DNA encoding the yeastpre-pro-alpha leader sequence is amplified from yeast genomic DNA in aPCR reaction using one primer containing nucleotides 1-20 of the alphamating factor gene and another primer complementary to nucleotides255-235 of this gene (Kurjan and Herskowitz, Cell, 30: 933-43 (1982)).The pre-pro-alpha leader coding sequence and the novel compound's codingsequence fragments are ligated into a plasmid containing the yeastalcohol dehydrogenase (ADH2) promoter, such that the promoter directsexpression of a fusion protein consisting of the pre-pro-alpha factorfused to the mature novel compound. As taught by Rose and Broach, Meth.Enz. 185: 234-79, Goeddel ed., Academic Press, Inc., San Diego, Calif.(1990), the vector further includes an ADH2 transcription terminatordownstream of the cloning site, the yeast “2-micron” replication origin,the yeast leu-2d gene, the yeast REP1 and REP2 genes, the E. coliβ-lactamase gene, and an E. coli origin of replication. The β-lactamaseand leu-2d genes provide for selection in bacteria and yeast,respectively. The leu-2d gene also facilitates increased copy number ofthe plasmid in yeast to induce higher levels of expression. The REP1 andREP2 genes encode proteins involved in regulation of the plasmid copynumber.

The DNA construct described in the preceding paragraph is transformedinto yeast cells using a known method, e.g., lithium acetate treatment(Steams et al., Meth. Enz. 185: 280-97 (1990)). The ADH2 promoter isinduced upon exhaustion of glucose in the growth media (Price et al.,Gene 55: 287 (1987)). The pre-pro-alpha sequence effects secretion ofthe fusion protein from the cells. Concomitantly, the yeast KEX2 proteincleaves the pre-pro sequence from the mature compounds of the invention(Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330-4 (1984)).

Compounds of the invention may also be recombinantly expressed in yeastusing a commercially available expression system, e.g., the PichiaExpression System (Invitrogen, San Diego, Calif.), following themanufacturer's instructions. This system also relies on thepre-pro-alpha sequence to direct secretion, but transcription of theinsert is driven by the alcohol oxidase (AOX1) promoter upon inductionby methanol. The secreted novel compound is purified from the yeastgrowth medium by, e.g., the methods used to purify the novel compoundfrom bacterial and mammalian cell supernatants.

Alternatively, the DNA encoding the novel compounds may be cloned intothe baculovirus expression vector pVL1393 (PharMingen, San Diego,Calif.). This novel compound-containing vector is then used according tothe manufacturer's directions (PharMingen) to infect Spodopterafrugiperda cells in sF9 protein-free media and to produce recombinantprotein. The protein is purified and concentrated from the media using aheparin-Sepharose column (Pharmacia, Piscataway, N.J.) and sequentialmolecular sizing columns (Amicon, Beverly, Mass.), and resuspended inPBS. SDS-PAGE analysis shows a single band and confirms the size of theprotein, and Edman sequencing on a Proton 2090 Peptide Sequencerconfirms its N-terminal sequence.

For example, the DNA sequence encoding the desired novel compound may becloned into a plasmid containing a desired promoter and, optionally, aleader sequence (see, e.g., Better et al., Science 240: 1041-3 (1988)).The sequence of this construct may be confirmed by automated sequencing.The plasmid is then transformed into E. coli, strain MC1061, usingstandard procedures employing CaCl₂ incubation and heat shock treatmentof the bacteria (Sambrook et al., supra). The transformed bacteria aregrown in LB medium supplemented with carbenicillin, and production ofthe expressed protein is induced by growth in a suitable medium. Ifpresent, the leader sequence will affect secretion of the mature novelcompound and be cleaved during secretion. The secreted recombinantprotein is purified from the bacterial culture media by the methoddescribed herein below.

Alternatively, the polypeptides of the present invention may beexpressed in an insect system. Insect systems for protein expression arewell known to those of skill in the art. In one such system, Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes in Spodoptera frugiperda cells or in Trichoplusialarvae. The novel compound's coding sequence is cloned into anonessential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofthe novel compound will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein coat. The recombinant viruses arethen used to infect S. frugiperda cells or Trichoplusia larvae in whichthe polypeptide is expressed (Smith et al., J. Virol. 46: 584 (1983);Engelhard et al., Proc. Natl. Acad. Sci. USA 91: 3224-7 (1994)).

In another example, the DNA sequence encoding the novel compound may beamplified by PCR and cloned into an appropriate vector, for example,pGEX-3X (Pharmacia, Piscataway, N.J.). The pGEX vector is designed toproduce a fusion protein comprising glutathione-S-transferase (GST),encoded by the vector, and a protein encoded by a DNA fragment insertedinto the vector's cloning site. The primers for the PCR may be generatedto include, for example, an appropriate cleavage site. The recombinantfusion protein may then be cleaved from the GST portion of the fusionprotein. The pGEX-3X/PYY analog polypeptide construct is transformedinto E. coli XL-1 Blue cells (Stratagene, La Jolla, Calif.), andindividual transformants are isolated and grown at 37° C. in LB medium(supplemented with carbenicillin) to an optical density at wavelength600 nm of 0.4, followed by further incubation for 4 hours in thepresence of 0.5 mM Isopropyl β-D-Thiogalactopyranoside (Sigma ChemicalCo., St. Louis, Mo.). Plasmid DNA from individual transformants ispurified and partially sequenced using an automated sequencer to confirmthe presence of the desired gene insert in the proper orientation.

The fusion protein, expected to be produced as an insoluble inclusionbody in the bacteria, may be purified as follows. Cells are harvested bycentrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; andtreated with 0.1 mg/mL lysozyme (Sigma Chemical Co.) for 15 min. at roomtemperature. The lysate is cleared by sonication, and cell debris ispelleted by centrifugation for 10 min. at 12,000×g. The fusionprotein-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mMEDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000×g.The pellet is resuspended in standard phosphate buffered saline solution(PBS) free of Mg⁺⁺ and Ca⁺⁺. The fusion protein is further purified byfractionating the resuspended pellet in a denaturing SDS polyacrylamidegel (Sambrook et al., supra). The gel is soaked in 0.4 M KCl tovisualize the protein, which is excised and electroeluted in gel-runningbuffer lacking SDS. If the GST/novel compound fusion protein is producedin bacteria as a soluble protein, it may be purified using the GSTPurification Module (Pharmacia Biotech).

The fusion protein may be subjected to digestion to cleave the GST fromthe mature novel protein. The digestion reaction (20-40 μg fusionprotein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 mL PBS) isincubated 16-48 hrs. at room temperature and loaded on a denaturingSDS-PAGE gel to fractionate the reaction products. The gel is soaked in0.4 M KCl to visualize the protein bands. The identity of the proteinband corresponding to the expected molecular weight of the novelcompound may be confirmed by partial amino acid sequence analysis usingan automated sequencer (Applied Biosystems Model 473A, Foster City,Calif.).

In a particularly preferred method of recombinant expression of thenovel compounds, 293 cells may be co-transfected with plasmidscontaining the DNA of the novel compound in the pCMV vector (5′ CMVpromoter, 3′ HGH poly A sequence) and pSV2neo (containing the neoresistance gene) by the calcium phosphate method. Preferably, thevectors should be linearized with ScaI prior to transfection. Similarly,an alternative construct using a similar pCMV vector with the neo geneincorporated can be used. Stable cell lines are selected from singlecell clones by limiting dilution in growth media containing 0.5 mg/mLG418 (neomycin-like antibiotic) for 10-14 days. Cell lines are screenedfor expression of the novel compound by ELISA or Western blot, andhigh-expressing cell lines are expanded for large scale growth.

It is preferable that the transformed cells are used for long-term,high-yield protein production and as such stable expression isdesirable. Once such cells are transformed with vectors that containselectable markers along with the desired expression cassette, the cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The selectable marker is designed to conferresistance to selection, and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell.

A number of selection systems may be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk−, hgprt− or aprt− cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside, G418; also, that confers resistance tochlorsulfuron; and hygro, that confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, β-glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin.

Many of the novel compounds of the present invention may be producedusing a combination of both automated peptide synthesis and recombinanttechniques. For example, a compound of the invention may contain acombination of modifications including deletion, substitution, andinsertion by PEGylation. Such compound may be produced in stages. In thefirst stage, an intermediate form of the novel compound containing themodifications of deletion, substitution, insertion, and any combinationthereof, may be produced by recombinant techniques as described. Thenafter an optional purification step as described below, the intermediatepolypeptide is PEGylated through chemical modification with anappropriate PEGylating reagent (e.g., from Nectar TransformingTherapeutics, San Carlos, Calif.) to yield the desired compound. Oneskilled in the art will appreciate that the above-described proceduremay be generalized to apply to the novel compound containing acombination of modifications selected from deletion, substitution,insertion, derivation, and other means of modification well known in theart and contemplated by the present invention.

It may be desirable to purify the novel compounds generated by thepresent invention. Peptide purification techniques are well known tothose of skill in the art. These techniques involve, at one level, thecrude fractionation of the cellular milieu to polypeptide andnon-polypeptide fractions. Having separated the polypeptide from otherproteins, the polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography, polyacrylamidegel electrophoresis, and isoelectric focusing. A particularly efficientmethod of purifying peptides is reverse phase HPLC, followed bycharacterization of purified product by liquid chromatography/massspectrometry (LC/MS) and Matrix-Assisted Laser Desorption Ionization(MALDI) mass spectrometry. Additional confirmation of purity is obtainedby determining amino acid analysis.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. Various techniques suitable for use in peptidepurification will be well known to those of skill in the art. Theseinclude, for example, precipitation with ammonium sulphate, PEG,antibodies, and the like; heat denaturation, followed by centrifugation;chromatography steps such as ion exchange, gel filtration, reversephase, hydroxylapatite and affinity chromatography; isoelectricfocusing; gel electrophoresis; and combinations of such and othertechniques. As is generally known in the art, it is believed that theorder of conducting the various purification steps may be changed, orthat certain steps may be omitted, and still result in a suitable methodfor the preparation of a substantially purified protein or peptide.Methods for purifying a polypeptide can be found in U.S. Pat. No.5,849,883. These documents describe specific exemplary methods for theisolation and purification of G-CSF compositions that may be useful inisolating and purifying the novel compounds of the invention. Given thedisclosure of these patents, it is evident that one of skill in the artwould be well aware of numerous purification techniques that may be usedto purify polypeptides from a given source. Also it is contemplated thata combination of anion exchange and immunoaffinity chromatography may beemployed to produce purified compounds of the invention.

Accordingly, the phrase “isolated polypeptide or peptide” refers to apolypeptide or peptide that is substantially free of cellular materialor other contaminating proteins from the cell or tissue source fromwhich the protein is derived, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus,protein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, or 5% (bydry weight) of heterologous protein (also referred to herein as a“contaminating protein”). When the protein, peptide, or fragment thereofis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest. Inpreferred embodiments, purified or isolated preparations will lack anycontaminating proteins from the same animal from which the protein isnormally produced, as can be accomplished by recombinant expression of,for example, a human protein in a non-human cell.

Certain preferred methods for synthesis are described in thecommonly-assigned U.S. Pat. No. 454,533 (file Dec. 6, 1999) the entiretyof which is incorporated herein by reference.

For all indications, the novel compounds may be administeredperipherally at a dose of about 1 μg to about 5 mg per day in single ordivided doses or controlled continual release, or at about 0.01 μg/kg toabout 500 μg/kg per dose, more preferably about 0.05 μg/kg to about 250μg/kg, most preferably below about 50 μg/kg. Doses may be administeredone, two, three or four times a day. Dosages in these ranges will varywith the potency of each analog or derivative, of course, and may bedetermined by one of skill in the art.

In the methods of the present invention, the polypeptides may beadministered separately or together with one or more other compounds andcompositions that exhibit a long term or short-term action to reducenutrient availability, including, but not limited to other compounds andcompositions that comprise an amylin or amylin analog agonist, salmoncalcitonin or salmone calcitonin agonist, a cholecystokinin (CCK) or CCKagonist, a leptin (OB protein) or leptin agonist, an exendin or exendinanalog agonist, or a GLP-1 or GLP-1 analog agonist or a PYY or PYYanalog, or a PYY related polypeptide. Suitable amylin agonists include,for example, [25,28,29 Pro-]-human amylin (also known as “pramlintide,”and described in U.S. Pat. Nos. 5,686,511 and 5,998,367). The CCK usedis preferably CCK octopeptide (CCK-8). Leptin is discussed in, forexample, (Pelleymounter, Cullen et al., Science 269: 540-543 (1995);Halaas, Gajiwala et al., Science 269: 543-6 (1995); Campfield, Smith etal., Science 269: 546-549 (1995)). Suitable exendins include exendin-3and exendin-4, and exendin agonist compounds include, for example, thosedescribed in PCT Publications WO 99/07404, WO 99/25727, and WO 99/25728.Suitable PYY polypeptides and analogs include those described in U.S.application Ser. Nos. 11/055,093 and 11/055,098.

Pharmaceutical Compositions

The present invention also relates to pharmaceutical compositionscomprising a therapeutically or prophylactically effective amount of atleast one compound of the invention, or a pharmaceutically acceptablesalt thereof, together with pharmaceutically acceptable diluents,preservatives, solubilizers, emulsifiers, adjuvants and/or carriersuseful in the delivery of the novel compounds. Such compositions mayinclude diluents of various buffer content (e.g., acetate, citrate,tartrate, phosphate, TRIS), pH and ionic strength; additives such assurfactants and solubilizing agents (e.g., sorbitan monooleate,lecithin, Pluronics, Tween 20 & 80, Polysorbate 20 & 80, propyleneglycol, ethanol, PEG-40, sodium dodecyl sulfate), anti-oxidants (e.g.,monothioglyercol, ascorbic acid, acetylcysteine, sulfurous acid salts(bisulfise and metabisulfite), preservatives (e.g., phenol, meta-cresol,benzyl alcohol, parabens (methyl, propyl, butyl), benzalkonium chloride,chlorobutanol, thimersol, phenylmercuric salts, (acetate, borate,nitrate), and tonicity/bulking agents (glycerine, sodium chloride,mannitol, sucrose, trehalose, dextrose) incorporation of the materialinto particulate preparations of polymeric compounds, such as polylacticacid, polyglycolic acid, etc., or in association with liposomes. Suchcompositions will influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the present compounds.See, e.g., Remington's Pharmaceutical Sciences 1435-712, 18th ed., MackPublishing Co., Easton, Pa. (1990).

In general, the present compounds will be useful in the same way thatamylin is useful in view of their pharmacological properties. Onepreferred use is to peripherally administer such novel compounds for thetreatment or prevention of metabolic conditions and disorders. Inparticular, the compounds of the invention possess activity as agents toreduce nutrient availability, reduce food intake, and effect weightloss.

The novel compounds may be formulated for peripheral administration,including formulation for injection, oral administration, nasaladministration, pulmonary administration, topical administration, orother types of administration as one skilled in the art will recognize.Examples of formulations can be found in U.S. Pat. No. 6,410,511 andpatent application Ser. No. 10/159,779, incorporated herein by referencein their entirety. More particularly, administration of thepharmaceutical compositions according to the present invention may bevia any common route so long as the target tissue is available via thatroute. In a preferred embodiment, the pharmaceutical compositions may beintroduced into the subject by any conventional peripheral method, e.g.,by intravenous, intradermal, intramusclar, intramammary,intraperitoneal, intrathecal, retrobulbar, intrapulmonary (e.g., termrelease); by oral, sublingual, nasal, anal, vaginal, or transdermaldelivery, or by surgical implantation at a particular site. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time. Controlled continual release of the compositions of thepresent invention is also contemplated. Examples of microspheretechnology can be found in U.S. Pat. No. 6,458,387 and U.S. Pat. No.5,578,708, incorporated herein by reference in their entirety.

The formulation may be liquid or may be solid, such as lyophilized, forreconstitution. Aqueous compositions of the present invention comprisean effective amount of the novel compounds, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions. In some cases, it will beconvenient to provide a compound of the invention and anotherfood-intake-reducing, plasma glucose-lowering or plasma lipid-alteringagent, such as an amylin, an amylin agonist analog, a CCK or CCKagonist, or a leptin or leptin agonist, or an exendin or exendin agonistanalog, or a PYY or a PYY analog, in a single composition or solutionfor administration together. In other cases, it may be more advantageousto administer the additional agent separately from the novel compound.

The compounds of the invention may be prepared for administration assolutions of free base, or pharmacologically acceptable salts in watersuitably mixed with a surfactant, such as hydroxypropylcellulose. Asused herein, the phrase “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable, preferably nontoxic,acids and bases, including inorganic and organic acids and bases,including but not limited to, sulfuric, citric, maleic, acetic, oxalic,hydrochloride, hydro bromide, hydro iodide, nitrate, sulfate, bisulfite,phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate,citrate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-nap-hthoate)) salts.Pharmaceutically acceptable salts include those formed with free aminogroups such as, but not limited to, those derived from hydrochloric,phosphoric, acetic, oxalic, and tartaric acids. Pharmaceuticallyacceptable salts also include those formed with free carboxyl groupssuch as, but not limited to, those derived from sodium, potassium,ammonium, sodium lithium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, and procaine. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

In one embodiment, the pharmaceutical compositions of the presentinvention are formulated so as to be suitable for parenteraladministration, e.g., via injection or infusion. Preferably, the novelcompound is suspended in an aqueous carrier, for example, in an isotonicbuffer solution at a pH of about 3.0 to about 8.0, preferably at a pH ofabout 3.0 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffersinclude sodium acetate/acetic acid, sodium lactate/lactic acid, ascorbicacid, sodium citrate-citric acid, sodium bicarbonate/carbonic acid,sodium succinate/succinic acid, histidine, Sodium benzoate/benzoic acid,and sodium phosphates, and Tris(hydroxymethyl)aminomehane. A form ofrepository or “depot” slow release preparation may be used so thattherapeutically effective amounts of the preparation are delivered intothe bloodstream over many hours or days following transdermal injectionor delivery.

The pharmaceutical compositions suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and should befluid that is easily syringable. It is also desirable for the PPFpolypeptide of the invention to be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., sorbitol, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), dimethylacetamide, cremorphor EL,suitable mixtures thereof, and oils (e.g., soybean, sesame, castor,cottonseed, ethyl oleate, isopropyl myristate, glycofurol, corn). Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. The prevention ofthe action of microorganisms can be brought about by variousantibacterial an antifungal agents, for example, meta-cresol, benzylalcohol, parabens (methyl, propyl, butyl), chlorobutanol, phenol,phenylmercuric salts (acetate, borate, nitrate), sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude tonicity agents (for example, sugars, sodium chloride).Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption (forexample, aluminum monostearate and gelatin). An exemplary pharmaceuticalcomposition may be 0.1 to 5% compound of the invention in an aqueoussystem along with approximately 0.02 to about 0.5% (w/v) of an acetate,phosphate, citrate, or glutamate buffer to a pH of the final compositionof approximately 3.0 to about 6.0 as well as approximately 1.0 to 10%(w/v) of a carbohydrate or polyhydric alcohol tonicifier; and,optionally, approximately 0.005 to 1.0% (w/v) of a preservative selectedfrom the group consisting of m-cresol, benzyl alcohol, parabens andphenol.

Sterile injectable solutions may be prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum-drying and freeze-drying techniquesthat yield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Generally, a therapeutically or prophylactically effective amount of thepresent novel compounds will be determined by the age, weight, andcondition or severity of the diseases or metabolic conditions ordisorders of the recipient. See, e.g., Remington's PharmaceuticalSciences 697-773. See also Wang and Hanson, Parenteral Formulations ofProteins and Peptides: Stability and Stabilizers, Journal of ParenteralScience and Technology, Technical Report No. 10, Supp. 42:2 S (1988).Typically, a dosage of between about 0.001 μg/kg body weight/day toabout 1000 μg/kg body weight/day, may be used, but more or less, as askilled practitioner will recognize, may be used. Dosing may be one ormore times daily, or less frequently, and may be in conjunction withother compositions as described herein. It should be noted that thepresent invention is not limited to the dosages recited herein.

Appropriate dosages may be ascertained through the use of establishedassays for determining level of metabolic conditions or disorders inconjunction with relevant dose-response data. The final dosage regimenwill be determined by the attending physician, considering factors thatmodify the action of drugs, e.g., the drug's specific activity, severityof the damage and the responsiveness of the patient, the age, condition,body weight, sex and diet of the patient, the severity of any infection,time of administration and other clinical factors. As studies areconducted, further information will emerge regarding appropriate dosagelevels and duration of treatment for specific diseases and conditions.

An effective dose will typically be in the range of about 1 to 30 μg toabout 5 mg/day, preferably about 10 to 30 μg to about 2 mg/day and morepreferably about 5 to 100 μg to about 1 mg/day, most preferably about 5μg to about 500 μg/day, administered in a single or divided doses. Thedosages may be between about 0.01 to about 500 μg/dose. It iscontemplated that compounds of the invention can be administered 1, 2,3, 4 or more times a day. Accordingly, exemplary doses can be derivedfrom the total amount of drug to be given a day and the number dosesadministered a day. For example, exemplary doses can range from about0.125 μg/dose (0.5 μg given four times a day) to about 5 mg/dose (5 mggiven once a day). Other dosages may be between about 0.01 to about 100μg/kg/dose. Still other exemplary doses may be 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200μg/dose. The exact dose to be administered may be determined by one ofskill in the art and is dependent upon the potency of the particularcompound, as well as upon the age, weight and condition of theindividual. Administration should begin whenever the suppression ofnutrient availability, food intake, weight, blood glucose or plasmalipid lowering is desired, for example, at the first sign of symptoms orshortly after diagnosis of obesity, diabetes mellitus, orinsulin-resistance syndrome. Administration may be by any route, e.g.,injection, preferably subcutaneous or intramuscular, oral, nasal,transdermal, etc. Dosages for certain routes, for example oraladministration, may be increased to account for decreasedbioavailablity, for example, by about 5-100 fold.

In one embodiment, where the pharmaceutical formulation is to beadministered parenterally, the composition is formulation so as todeliver a dose of the novel compounds ranging from 0.1 μg/kg to 100mg/kg body weight/day, preferably at doses ranging from 10 μg/kg toabout 50 mg/kg body weight/day. Parenteral administration may be carriedout with an initial bolus followed by continuous infusion to maintaintherapeutic circulating levels of drug product. Those of ordinary skillin the art will readily optimize effective dosages and administrationregimens as determined by good medical practice and the clinicalcondition of the individual patient.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe agents and the routes of administration. The optimal pharmaceuticalformulation will be determined by one of skill in the art depending onthe route of administration and the desired dosage. See, e.g.,Remington's Pharmaceutical Sciences, supra, pages 1435-1712. Suchformulations may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the administered agents.Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface areas or organ size.Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation, especially in light of thedosage information and assays disclosed herein, as well as thepharmacokinetic data observed in animals or human clinical trials.

It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention may be useful in fields of humanmedicine and veterinary medicine. Thus the subject to be treated may bea mammal, preferably human or other animal. For veterinary purposes,subjects include for example, farm animals including cows, sheep, pigs,horses and goats, companion animals such as dogs and cats, exotic and/orzoo animals, laboratory animals including mice, rats, rabbits, guineapigs and hamsters; and poultry such as chickens, turkeys, ducks andgeese.

To assist in understanding the present invention, the following Examplesare included. The experiments relating to this invention should not, ofcourse, be construed as specifically limiting the invention and suchvariations of the invention, now known or later developed, which wouldbe within the purview of one skilled in the art are considered to fallwithin the scope of the invention as described herein and hereinafterclaimed.

EXAMPLES Example 1 Synthesis of the Caloric Intake Lowering Polypeptides

The following polypeptides can be synthesized using standard polypeptidesynthesis methods. Such methods are described below and in U.S. Pat. No.6,610,824 and U.S. Pat. No. 5,686,411 and in U.S. Pat. No. 454,533(filed Dec. 6, 1999), the entireties of which are incorporated herein byreference.

The polypeptides are assembled on 4-(2′-4′-dimethoxyphenyl)-Fmocaminomethyl phenoxy acetamide norleucine MBHA resin (Novabiochem, 0.55mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.). Ingeneral, single-coupling cycles are used throughout the synthesis andFast Moc (HBTU activation) chemistry is employed. However, at somepositions coupling may be less efficient than expected and doublecouplings required. Deprotection (Fmoc group removal) of the growingpeptide chain using piperidine likewise may not always be efficient andrequire double deprotection. Final deprotection of the completed peptideresin is achieved using a mixture of triethylsilane (0.2 mL),ethanedithiol (0.2 mL), anisole (0.2 mL), water (0.2 mL) andtrifluoroacetic acid (15 mL) according to standard methods (Introductionto Cleavage Techniques, Applied Biosystems, Inc.) The peptides areprecipitated in ether/water (50 mL) and centrifuged. The precipitate isreconstituted in glacial acetic acid and lyophilized. The lyophilizedpeptides are dissolved in water). Crude purity is then determined.

Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACN) are usedin purification and analysis steps.

Solutions containing the various polypeptides are applied to apreparative C-18 column and purified (10% to 40% Solvent B in Solvent Aover 40 minutes). Purity of fractions are determined isocratically usinga C-18 analytical column. Pure fractions are pooled furnishing theabove-identified peptide. Analytical RP-HPLC (gradient 30% to 60%Solvent B in Solvent A over 30 minutes) of the lyophilized peptide todetermine retention time.

Example 2 Receptor Binding Assays

Initially, polypeptides can be used in assays to determine bindingability to amylin, calictonin and CGRP receptors. Binding assays fordetermining interactions with the amylin-receptor, thecalcitonin-receptor, and the CGRP receptor are described for example inU.S. Pat. No. 5,264,372, the entirety of which is incorporated herein byreference.

In more detail, evaluation of the binding of compounds of the inventionto amylin receptors can be carried out as follows. ¹²⁵I-rat amylin(Bolton-Hunter labeled at the N-terminal lysine) is purchased fromAmersham Corporation (Arlington Heights, Ill.). Unlabeled peptides areobtained from BACHEM Inc. (Torrance, Calif.) and Peninsula Laboratories(Belmont, Calif.).

Male Sprague-Dawley® rats (200-250) grams are sacrificed bydecapitation. Brains are removed to cold phosphate-buffered saline(PBS). From the ventral surface, cuts are made rostral to thehypothalamus, bounded laterally by the olfactory tracts and extending ata 45 angle medially from these tracts. This basal forebrain tissue,containing the nucleus accumbens and surrounding regions, is weighed andhomogenized in ice-cold 20 mM HEPES buffer (20 mM HEPES acid, pHadjusted to 7.4 with NaOH at 23 C). Membranes are washed three times infresh buffer by centrifugation for 15 minutes at 48,000.times.g. Thefinal membrane pellet is resuspended in 20 mM HEPES buffer containing0.2 mM phenylmethylsulfonyl fluoride (PMSF).

To measure ¹²⁵I-amylin binding, membranes from 4 mg original wet weightof tissue are incubated with ¹²⁵I-amylin at 12-16 pM in 20 mM HEPESbuffer containing 0.5 mg/ml bacitracin, 0.5 mg/ml bovine serum albumin,and 0.2 mM PMSF. Solutions are incubated for 60 minutes at 2 C.Incubations are terminated by filtration through GF/B glass fiberfilters (Whatman Inc., Clifton, N.J.) which has been presoaked for 4hours in 0.3% poylethyleneimine in order to reduce nonspecific bindingof radiolabeled peptides. Filters are washed immediately beforefiltration with 5 ml cold PBS, and immediately after filtration with 15ml cold PBS. Filters are removed and radioactivity assessed in agamma-counter at a counting efficiency of 77%. Competition curves aregenerated by measuring binding in the presence of 10⁻¹² to 10⁻⁶ Munlabeled test compound and are analyzed by nonlinear regression using a4-parameter logistic equation (Inplot program; GraphPAD Software, SanDiego).

In this assay, purified human amylin binds to its receptor at a measuredIC₅₀ of about 50 pM. Results for test compounds of the invention are setforth in the table below, showing that each of the compounds hassignificant receptor binding activity.

Evaluation of the binding of compounds of the invention to CGRPreceptors was essentially as described for amylin except using 125IhCGRP and membranes prepared from SK-N-MC cells, known to express CGRPreceptors (Muff, R. et. al. Ann NY Acad. Sci. 1992: 657, 106-16).Binding assays were performed as described for amylin except using13,500 cpm 125I-hCGRP/well or 21.7 pM/well (Amersham).

Binding to the calcitonin receptor may be investigated using CHO cellsor T47D cells, which also express the calcitonin receptor (Muff R. et.al, Ann NY Acad Sci. 1992, 657:106-16 and Kuestner R. E. et. al. MolPharmacol. 1994, 46:246-55), as known in the art

TABLE 2 EC₅₀ values (nM) for polypeptides Compound Amylin CalcitoninCGRP 1 0.028 0.029 2.342 2 0.047 0.052 33.988 3 0.023 0.020 0.490 40.035 0.019 8.500 5 0.022 0.018 2.600 6 0.030 nt nt 7 0.057 nt 7.540 88.070 0.478 175.665 9 0.043 0.014 1.600 nt denotes not tested

Example 3 Activity of Polypeptides on Food Intake

Female NIH/Swiss mice (8-14 weeks old) were group housed with a 12:12hour light:dark cycle. Water and a standard pelleted mouse chow diet areavailable ad libitum, except as noted. Animals are fasted starting atapproximately 1500 hrs, 1 day prior to experiment.

At time=0 min, all animals are given an intraperitoneal injection ofvehicle or polypeptide in a volume of 200 uL/mouse and immediately givena pre-weighed amount (10-15 g) of the standard chow. Food is removed andweighed at 30, 60, 120 and 180 minutes to determine the amount of foodconsumed. The effects of treatment on food intake are expressed as %change relative to control.

As can be seen in FIG. 1, Compound 2, at doses from 25-300 nmol/kg,dose-dependently reduced food intake at 30 minutes post injection. Thetable below depicts reduced food intake with polypeptides administeredperipherally (intraperitoneal injection) at doses 25 nmol/kg. The dataat time points 30, 60, 120, and 180 minutes represents the percentdecrease in cumulative food intake compared to the vehicle.

TABLE 3 Compound (SEQ ID NO:) 30 min 60 min 120 min 180 min  1 (137) −58−46 −33 −22  2 (136) −58 −54 −52 nt  3 (135) −58 −52 −37 −33  4 (40) −42−31 −35 −30  5 (43) −66 −53 −29 −27  6 −48 −45 −23 nt  7 (134) −60 −52−23 nt  8 −6 −15 −25 −28  9 (71) −80 −64 −43 nt  10 −19 −20 −35 nt 11(41) −52 −47 −38 −35 12 (42) −43 −39 −37 −32 13 (44) −40 −33 −25 −24 14(45) −52 −36 −28 −33 15 (46) −67 −59 −37 −30  16 −26 −29 −30 −27 17 (47)−42 −30 −30 −25 18 (48) −2 −7 −16 −21 19 (49) −25 −25 −35 −31 20 (50) −9−21 −30 −31 21 (51) 9 −5 −18 −18 22 (52) −11 −20 −31 −30 23 (53) 8 0 −19−12 24 (54) −40 −34 −35 −35 25 (55) −29 −34 −45 nt 26 (56) −29 −36 −47nt 27 (57) −12 −11 −32 nt 28 (58) −8 −16 −28 nt 29 (59) 4 −1 −25 nt 30(60) −1 −2 −19 nt 31 (61) −11 −18 −23 nt 32 (62) −15 −21 −31 nt  33 −7−10 −15 nt  34 −11 −6 −16 nt 35 (63) −20 −16 −18 nt 36 (64) −34 −22 −24−25 37 (65) −3 −2 −16 nt  38 −24 −13 −8 nt 39 (66) 7 −14 −23 nt  40 −11−5 −2 nt  41 −4 −9 −12 nt  42 −11 −18 −32 nt  43 −4 −7 −18 nt 44 (69) −6−13 −25 nt  45 −13 −7 −3 nt  46 −6 −11 −16 nt 47 (70) −5 −13 −27 nt 48(72) −54 −51 −36 nt 49 (73) −33 −26 −25 nt 50 (74) −70 −62 −48 nt 51(75) −44 −39 −35 nt 52 (76) −29 −24 −23 nt 53 (77) −92 −89 −36 nt  54 1−4 −10 nt  55 9 −5 −12 nt  56 4 −13 −16 nt 57 (78) −18 −24 −23 nt 58(79) −62 −51 −29 nt 59 (80) −81 −77 −50 nt 60 (81) −43 −40 −26 nt 61(82) −23 −27 −32 nt 62 (83) −14 −22 −38 nt 63 (84) −19 −22 −28 nt 64(85) −65 −58 −44 nt 65 (86) −33 −29 −32 nt 66 (87) −13 −15 −28 nt  67−10 −11 −12 nt 68 (88) −10 −13 −21 nt 69 (89) −29 −31 −45 nt 70 (90) −76−64 −47 nt 71 (91) −7 −13 −22 −18 72 (92) 0 −8 −13 −19 73 (93) −51 −31−23 −28 74 (94) −42 −32 −31 nt 75 (95) −60 −52 −38 nt 76 (96) −25 −29−40 nt 77 (97) −46 −43 −44 nt 78 (98) −57 −44 −44 nt 79 (99) −49 −40 −33nt  80 (100) −32 −28 −22 nt  81 (101) −28 −24 −33 nt  82 (102) −7 −13−16 −19  83 (103) −7 −13 −22 −12  84 (104) −53 −40 −20 nt  85 (105) 3−16 −16 nt  86 (106) −44 −26 −16 nt  87 (107) −43 −32 −21 nt  88 (108)−64 −61 −39 −22  89 (109) −6 −13 −22 −20  90 (110) −55 −41 −24 −15  91(111) −59 −47 −26 −24  92 (112) −31 −29 −30 −27  93 (113) −43 −30 −27−29  94 (114) −62 −42 −36 −31  95 (115) −81 −69 −34 −31  96 (116) −49−38 −19 −23  97 (117) −78 −76 −60 −40  98 −18 −13 −5  −1  99 (118) −57−55 −50 nt 100 (119) −60 −52 −41 nt 101 (120) −52 −48 −35 nt 102 (121)−58 −53 −45 nt 103 (122) −50 −44 −30 nt 104 (123) −69 −67 −54 nt 105(124) −83 −82 −52 nt 106 (125) −58 −54 −39 nt 107 (126) −84 −78 −47 nt108 (127) −70 −66 −38 nt 109 (128) −61 −54 −43 nt 110 (129) −80 −72 −59nt 111 (130) −39 −37 −32 nt 112 (131) −62 −65 −50 nt 113 (132) −79 −86−55 nt 114 (133) −17 −20 −25 nt 115 6 −3 −25 −25 116 5 5 3 nt 117 −13−11 −3 nt 118 −4 0 13 nt 119 6 −8 −11 nt 120 −3 1 −6  −7 121 5 2 −1  3122 −6 −12 −23 −21 123 1 −13 −17 −13 124 4 −4 −15 −16 125 10 −1 −6 nt126 5 −10 −20 nt 127 −5 −14 −12 −12 nd = not done

Example 4 Activity of Compounds of the Invention on Weight Reduction andCaloric Intake

Individually housed male Sprague-Dawley® rats (350 g; 12-h light/darkcycle) were maintained on a high fat diet (58% kcal from fat) for 4weeks. At the end of fattening period, 14-day osmotic pumps (DurectCorp.) were implanted interscapularly under anesthesia. Rats receivedpumps continuously delivering vehicle (50% DMSO) or polypeptide at adose of 2.9 nmol/kg/day. Food intake and body weight measurements wereobtained weekly. FIGS. 2A and 2B show that the polypeptides Compound 3,Compound 4, or Compound 5 produced a decrease in caloric intake and bodyweight gain throughout the 14-day test period.

The table below presents the percent body weight loss at week 1 and 2for several compounds.

TABLE 4 Body weight loss after administration of exemplary compounds ofthe invention Compound Week 1 Week 2 Compound 1 8.3* 10.5* Compound 29.8* 9.4* Compound 3 5.9* 6.7* Compound 4 6.8* 9.2* Compound 5 8.6*11.3* Compound 6 2.9* 3.8* Compound 7 10.0* 11.4* Compound 8 2.3 2.5Compound 9 4.9* 4.9* *P < 0.05 compared to controls

Example 5 Body Composition

Individually housed male Sprague-Dawley® rats (420 g; 12-h light/darkcycle) were maintained on a high fat diet (58% kcal from fat) for 4weeks. At the end of fattening period, 14-day osmotic pumps (DurectCorp.) were implanted interscapularly under anesthesia. Rats receivedpumps that continuously delivered vehicle (50% DMSO) or Compound 1 at adose of 70 nmol/kg/day. Animals were sacrificed on Day 12. Carcasseswere immediately frozen and body composition (fat and protein) measuredby chemical analysis (Covance Laboratories, Madison, Wis.). FIG. 3 showsthat, as a percent of total body mass, fat content was reduced in ratstreated with Compound 1 compared to controls. In addition, Compound 1increased the percent of lean mass content.

Example 6 Gastric Emptying and Ion Calcium

Gastric emptying was monitored by measuring the appearance in plasma ofgavaged tritiated glucose. Subjects were conscious, male Sprague Dawley®rats (7-9 weeks of age, 12:12 h light:dark cycle) with ad libitum accessto food and water until the start of the experiment. Prior to dosing,food and water were removed. At t=−5 min, peptide or vehicle (200 μlsaline) was administered subcutaneously. At t=0 min, a solution of 1 mlsterile water containing 5 μCi D-[3-3H] glucose (Dupont, Wilmington,Del., USA) was given by oropharyngeal tube. At t=20 min, topicalanesthetic (Hurricaine®, 20% benzocaine liquid) was applied to the tipof the tail. At t=40 min, the tip of the tail was ligated with a scalpeland ˜250 μl blood was collected into heparinized tubes. Plasma was thenimmediately assayed for ionized calcium using a Ciba/Corning 634 Ca/pHanalyzer (Ciba/Corning, Inc., Medfield, Mass.). A 10 μl plasma samplewas pipetted into prepared scintillation vials (0.5 ml water+2 mlscintillation cocktail (Ecolite scintillation cocktail ICN, Costa Mesa,Calif.)), vortexed and counted in a β-counter (1209 Rack-beta;LKB-Wallac, Gaithersburg, Md.) for 1 minute/vial.

In FIG. 4A, points represent mean±sd of 6 SD rats (fed, conscious). Theindicated dose of peptide was injected subcutaneously at t=0. Blood wascollected 35 minutes later for cpm analysis. *All points p<0.001 vs.saline control; ANOVA, Dunnett's test. From non-linear regression:ED50=2.3 μg/kg. Bottom=25 cpm/10 μl; Top=328 cpm/10 μl. Maximum decreasein plasma cpm: −92%. Goodness of fit: r2=0.9992.

In FIG. 4B, points represent mean±sd of 6 SD rats (fed, conscious). Theindicated dose of peptide was injected subcutaneously at t=0. Blood wascollected 35 minutes later for cpm analysis. *P<0.001 vs. salinecontrol; ANOVA, Dunnett's test. From non-linear regression: ED50=1.1μg/kg. Bottom=1.2 mmol/L; Top=1.3 mmol/L. Maximum decrease in plasmaiCa: −14% Goodness of fit: r2=0.9936.

Example 7 Triglycerides

In study 1, female Harlan Sprague-Dawley® rats (HSD; HarlanTeklad,Indianapolis, Ind.) retired from breeding were fed a high-fat diet (40%of calories from fat; Diet #TD95217, HarlanTeklad) for 9 weeks prior tothe initiation of the study, continuing on this diet throughout theexperimental period. One group of rats were fed ad libitum throughoutthe study to examine the metabolic effects of Compound 1, arepresentative compound of the invention, treatment (n=10) relative tocontrol, untreated rats (n=10). To determine the metabolic effects ofCompound 1 in the setting of food restriction (“dieting”), a secondgroup of rats was given 75% of their pre-study baseline food intake eachday for 10 days prior to initiation of Compound 1 (n=10) or vehicle(n=10) treatment (˜5% body weight loss vs. pre-restriction body weight).At that time, all rats received Alzet® osmotic pumps (Durect, Cupertino,Calif.) surgically implanted in the intrascapular region subcutaneouslyunder isoflurane anesthesia. The pumps were prepared to deliver eitherCompound 1 (101 μg/kg/day) or vehicle (50% dimethoxysulfoxide [DMSO] inwater) continuously for 21 days. Food-restricted rats continued to begiven 75% of their baseline food intake each day. After 21 days oftreatment, blood samples were obtained into heparinized syringes viacardiac puncture from isoflurane-anesthetized postabsorptive rats (˜2-4hr fasted in the a.m.) and plasma obtained for analyte analysis. Plasmatriglycerides were determined using standard automated analysis forclinical chemistry (LabCorp, Inc., San Diego, Calif.).

In study 2, male rats from the obesity-prone Levin strain of HSD rats(Charles River, Wilmington, Mass.) were fed a high-fat diet (32% ofcalories from fat; Diet #12266B, Research Diets, Inc., New Brunswick,N.J.) for 6 wk prior to initiation of the study, and continued on thisdiet throughout the experiment. Initiation of treatment commenced withplacement of subcutaneous osmotic pumps containing Compound 1 or DMSOvehicle as per Study 1, and continued for 3 weeks (21 days). Metabolicparameters were compared between Compound 1-treated rats (303 μg/kg/day;n=10), vehicle-treated controls (n=10), and a pair-fed group (n=10)which received daily the amount of food eaten by Compound 1-treatedrats. Blood collection and analysis of plasma triglyceride concentrationwere performed as in Study 1, but using tail vein collection at the 1week and 2 week points.

Study 3 was conducted as per Study 2 except animals were treated for 8weeks (56 days) and at different doses of Compound 1. At 28 days oftreatment, fresh osmotic pumps replaced used pumps. The pumps wereprepared to deliver Compound 1 at doses of 1, 3, 10, or 100 μg/kg/day(n=10/group) or DMSO vehicle (controls, n=10). An additional pair-fedgroup (n=10) received vehicle and were fed daily the amount of foodeaten by the 100 μg/kg/day Compound 1 group. Blood collection andanalysis of plasma triglyceride concentration for baseline, 2, 4, and 6week points were obtained from conscious fed rats via tail veincollection into heparinized tubes; blood collection and analysis ofplasma triglyceride concentration for 8 week point was obtained fromisoflurane-anesthetized fed rats into heparinized syringes via cardiacpuncture, employing a standard triglyceride assay (Cobas Mira Plus,Roche Diagnostics).

Differences between mean plasma triglyceride levels across treatmentsfor a given timepoint of the study were evaluated one-way analysis ofvariance (ANOVA), followed by a Dunnett's Multiple Comparison Test(Prism v. 4.01, GraphPad Software, San Diego, Calif.). Differences wereconsidered statistically significant at p<0.05.

As indicated in FIGS. 5 to 7E, studies 1, 2, and 3, respectively,treatment with Compound 1 over 1-8 weeks resulted in significantly lowertriglyceride concentrations. FIG. 5 shows plasma triglycerideconcentration in Female Retired Breeder rats following a 21 daytreatment with Compound 1 via continuous subcutaneous (s.c.) infusion.Rats administered Compound 1 displayed significantly lower triglyceridelevels compared to ad lib fed controls: this effect was observedregardless of whether Compound 1 was given under ad lib fed conditionsor under food-restricted conditions. *p<0.05, **p<0.01 compared to adlib, vehicle-treated controls.

FIGS. 6A-6C depict plasma triglyceride concentration in Diet-inducedObese (DIO) Male Levin rats during a 3 week treatment with Compound 1via continuous s.c. infusion. Graphs represent values derived from bloodsamples collected at (A) 1 week, (B) 2 weeks, and (C) 3 weeks oftreatment. *p<0.05, **p<0.01 compared to vehicle-treated controls.

FIGS. 7A-7E show a dose response study of Compound 1, where doses as lowas 1 μg/kg/day resulted in a reduction in plasma triglycerideconcentrations when compared to vehicle controls at the 2, 4, 6 and 8week points (FIGS. 7B, C, D, and E, respectively). *p<0.05, **p<0.01compared to vehicle-treated controls. Treatment groups are indicated inthe legend.

Example 8 Ghrelin Assay

This example provides an exemplary assay for detecting the effect of thecompounds of the invention on ghrelin.

Male Harlan Sprague Dawley® (HSD) rats were housed at 22.8+/−0.8° C. ina 12:12 hour light:dark cycle. All experiments were performed in thelight cycle. Animals were fasted for approximately 20 hours beforeexperimentation. All animals were given free access to water until thestart of the experiment. The animals' tails were anesthetized with 20%benzocaine (Hurricaine, Beutlich Pharmaceutical, Waukegan, Ill.), andblood samples were collected from the tail vein. Total and activeghrelin concentrations were measured using Linco RIA kits GHRA-89HK andGHRA-88HK, respectively.

In study 1, HSD rats were subjected to periodic blood sampling from thetopically anesthetized tail and ghrelin levels were assayed. At t=0,rats (n=6) were injected s.c. with 125 μg/kg pentagastrin (Sigma) tostimulate gastric acid secretion (PG=0 min in FIG. 1), and 20 min laterwere injected subcutaneously (s.c.) with 10 μg rat amylin. The bloodsamples were analyzed for total and active (acylated) ghrelin (Linco).As shown in FIG. 8A, amylin reduced active ghrelin by ˜50% within 1hour.

Study 2 was conducted to examine whether exogenous amylin inhibitsghrelin secretion independent of pentagastrin stimulation. Fasted ratswere given either a subcutaneous injection of saline or of 30 μg/kg ratamylin or a subcutaneous injection of either saline or 125 μg/kgpentagastrin (Sigma, Lot#050K1525) at time=0 min. Rat amylin (AC0128,lot #AR2081-42A, Amylin Pharmaceuticals), at a dose of 30 μg/kg in 100μl of saline, or saline vehicle alone (n=5.5 respectively), was given bysubcutaneous injection at time 20 min. Blood plasma samples werecollected at least at times 0, 10, 20, 30, 60, and 90 min. Both FIGS. 8Band 8C show a reduction in total plasma ghrelin with the administrationof amylin compared to the saline control, and FIG. 8B confirms thatplasma ghrelin was reduced compared to the control in the presence ofamylin alone, i.e., without pentagastrin. Pentagastrin appears toenhance the ghrelin lowering effect of amylin.

Example 9 Pancreatic Function

Femoral artery and vein cannulations are performed in fasted,anesthetized HSD rats (weight 320-350 g) and they are allowed tostabilize for 90 minutes. At t=−30, an infusion of saline (1 ml/hr) orCompound 1 was started (1 ml/hr). At t=0, rats receive an imp injectionof caerulein, 10 μg/kg. Samples for plasma amylase, lipase werecollected at various time points from t=−30 to t=240

As shown in FIGS. 9A and 9B, treatment with Compound 1 attenuatedincreases in pancreatic enzyme activities in the blood in rat model ofacute pancreatitis suggesting that agonists of amylin may be perspectivedrug candidates for the prevention and treatment of pancreatitis.

Example 10 Gastric Acid Secretion of Amylin

Male Harlan Sprague Dawley® rats were housed at 22.8±0.8° C. in a 12:12hour light: dark cycle. The experiments were performed during the lightcycle. The rats, fed rat chow (Teklad L M 485, Madison, Wis.), werefasted for approximately 20 hours before experimentation. They weregiven water ad libitum until the start of the experiment.

The rats (age 9-14 weeks, body mass 264-395 g) were surgically fittedwith double gastric fistulae by the supplier (Zivic Miller, Catalognumber SCAO3.00). During laparotomy under halothane anesthesia, agrommet-shaped double lumen plug was sutured into the stomach wall. Twosilastic 2.3 mm internal diameter (entry and exit) cannulae connected tothe plug and communicating with the gastric lumen were tracked throughthe abdominal wall, subcutaneously to the interscapular region wherethey were separately exteriorized. The laparotomy wound was closed withclips, the rat was placed in a heated recovery cage for one day withfree access to water. Thereafter, the rats were housed singly with freeaccess to water and rat chow until they were subjected to overnightfasting for experiments which were performed on conscious rats, about10-15 days after surgery.

Gastric catheters were uncapped and attached to flexible PE240 tubingfor injection and sampling. To ensure patency of the catheters, 2-3 mLof saline solution at room temperature was injected and immediatelywithdrawn from the stomach. This was repeated until flow was easy andthe effluent was clean. Gastric acid secretion was measured at 10 minintervals by injecting 5 mL saline and 2 mL air via one catheter, thenimmediately withdrawing the same via the other catheter. In this wayerrors due to incomplete aspiration of the small, secreted volume couldbe minimized. Three mL of each gastric aspirate were titrated to pH 7.0with 0.01 N sodium hydroxide using a pH meter (Beckman model numberPHI34 Fullerton, Calif.). The amount of base required for eachtitration, corrected for the total volume aspirated, was used tocalculate the moles of acid in each sample.

After a baseline sample was collected and the recovered volume recorded,the animals were given a subcutaneous injection of 125 μg/kgpentagastrin (Peninsula Laboratories lot number 019945 and 034686), andthe 10 min gastric sampling continued for another 2 hours. At forty minafter pentagastrin injection, by which time a stable plateau of gastricacid secretion was observed, the rats were injected subcutaneously withsaline (n=6) or with rat amylin (batch number AR905-80, AmylinPharmaceuticals Inc., San Diego, Calif.) at doses of 0.01, 0.1, 1.0,10.0 or 100 μg (n=3, 3, 4, 5, 5 respectively).

As shown in FIG. 10A, pentagastrin stimulated gastric acid secretion4.6-fold from a basal rate of about 13.8±2.2 μmol/10 min to about63.4±3.3 μmol/10 min, 40 minutes after pentagastrin injection (grandmeans; P<0.0001). Amylin injected 40 min after pentagastrindose-dependently inhibited gastric acid production with half lives forthe onset of action of 8.6, 10.4, 5.8 and 6.3 min for doses of 0.1, 1,10 and 100 μg, respectively. With the highest (100 μg) dose of amylin,pentagastrin-stimulated secretion of acid was reduced by 93.4±2.6% onehour after amylin injection (P<0.001 for doses 0.1-100 μg). This rate ofsecretion was only 32% of the basal rate that preceded pentagastrininjection (P<0.01, t-test, Welch correction). The dose response foramylin inhibition of pentagastrin-stimulated acid secretion is shown inFIG. 10B. The ED₅₀ for the acid-suppressive effect of amylin was 0.05μg/rat±0.15 log units (41 pmol/kg).

Example 11 Gastroprotective Effects of Amylin

Fasted male rats, body weight 163-196 g, were injected subcutaneouslywith 0.1 mL saline (n=12) or with the same volume containing rat amylinat doses of 0.001, 0.01, 0.1, 0.3, 1, 3 or 10 μg (n=5, 5, 5, 9, 9, 5, 6respectively) 20 min before gavage with 1 mL absolute ethanol (ethylalcohol-200 proof dehydrated alcohol, USP, Spectrum Quality Products,Inc. Gardena, Calif.). Thirty min after gavage, each rat wasanesthetized with 5% halothane, the stomach excised, opened along thelesser curvature and everted to expose the mucosa. The everted stomachswere gently rinsed with saline and immediately graded for mucosal damageby each of 10 observers blinded to the experimental treatment. Thegrading scale was constrained between zero (no observable damage) and 5(100% of mucosal surface covered by hyperemia, ulceration, orsloughing), comparable to the 0-5 scoring system used by others, e.g.,Guidobono F. et al. Br J Pharmacol 120:581-586 (1997). FIG. 11A showsthe results of the injury score as a percent of the injury induced byethanol.

To determine whether a gastroprotective effect of amylin wasattributable to an amylin-specific mechanism, 4 rats were injectedintravenously with 3.0 mg of the selective amylin antagonist, AC187(Amylin Pharmaceuticals, Inc. San Diego, Calif.), 25 min before ethanolgavage, followed 5 min later (at t=−20 min from gavage) by 0.3 μg ratamylin injected subcutaneously. Stomachs were excised and graded forinjury 30 min after ethanol gavage, as described above. The result isprovided in the next to the last bar of FIG. 11A. The last bar shows theresults of injection of 3.0 mg of AC187 with no rat amylin injection. Amore selective look at the results are shown in FIG. 11B.

FIG. 11C shows that amylin, given as a subcutaneous injection 5 minbefore gavage with ethanol, dose-dependently protected the stomach frommucosal injury (P<0.05 with doses of 0.1 μg and higher). Amylin reducedthe injury score by 67% with doses of 0.3 μg and higher. The ED₅₀ forthe gastroprotective effect of amylin in this experimental system was0.036 μg (31 μmol/kg)±0.40 log units.

To further explore whether a gastroprotective effect of amylin couldoccur in response to glucose-stimulated secretion of endogenous amylin,the effect on ethanol-induced gastritis of prior intraperitoneal glucoseadministration (250 mg/0.5 mL D-glucose; t=−30 min from gavage; n=9) wascompared to the injury observed in vehicle-treated rats (n=23). Stomachswere excised and graded for injury 30 min after ethanol gavage, asdescribed above, and blood was taken for plasma glucose measurement. Theinjury response was also measured in glucose-treated rats coadministereda 3 mg intravenous bolus of AC187 (n=9).

Prior administration of D-glucose, which increased plasma glucose att=+30 min later to 123 mg/dL (vs 76 mg/dL in controls), and which hadpreviously been shown to increase endogenous plasma amylinconcentrations in fasted Sprague Dawley rats to 4.8±0.6 pM,significantly decreased gastric injury score by 18.5±4.6% (P<0.0005).However, pre-injection of AC187 had no effect on injury scores inglucose-treated rats.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1-17. (canceled)
 18. A method of treating eating disorders,insulin-resistance, obesity, abnormal postprandial hyperglycemia,diabetes of any kind, including Type I, Type II, and gestationaldiabetes, Metabolic Syndrome, Dumping Syndrome, hypertension,dyslipidemia, cardiovascular disease, hyperlipidemia, sleep apnea,cancer, pulmonary hypertension, cholecystitis, and osteoarthritiscomprising administering to a subject in need thereof an effectiveamount of a peptide having at least 92% sequence identity to the aminoacid sequence of SEQ ID NO:
 137. 19. The method of claim 18, wherein thepeptide having at least 92% sequence identity comprises the amino acidsequence of SEQ ID NO: 55, 67, 70, 73, 82, 83, 84, 89, 94, 100, 103,106, 107, 111, 112, 125, 133, or
 137. 20. The method of claim 18,wherein the peptide having at least 92% sequence identity comprises theamino acid sequence of SEQ ID NO:
 137. 21. The method of claim 18,wherein the peptide is linked to a polyethylene glycol polymer, apolyamino acid, or a fatty acid.
 22. A method of treating eatingdisorders, insulin-resistance, obesity, abnormal postprandialhyperglycemia, diabetes of any kind, including Type I, Type II, andgestational diabetes, Metabolic Syndrome, Dumping Syndrome,hypertension, dyslipidemia, cardiovascular disease, hyperlipidemia,sleep apnea, cancer, pulmonary hypertension, cholecystitis, andosteoarthritis comprising administering to a subject in need thereof aneffective amount of a peptide having at least 95% sequence identity tothe amino acid sequence of SEQ ID NO:
 137. 23. The method of claim 22,wherein the peptide having at least 95% sequence identity comprises theamino acid sequence of SEQ ID NO: 40, 42, 43, 46, 47, 48, 54, 64, 65,68, 69, 74, 78, 79, 80, 81, 85, 90, 91, 92, 93, 95, 96, 97, 98, 99, 101,102, 109, 118, 119, 121, 130, or
 137. 24. The method of claim 22,wherein the peptide having at least 95% sequence identity comprises theamino acid sequence of SEQ ID NO:
 137. 25. The method of claim 22,wherein the peptide is linked to a polyethylene glycol polymer, apolyamino acid, or a fatty acid.
 26. The method of claim 18 or claim 22,further comprising administering an amylin, [25,28,29 Pro-]-humanamylin, salmon calcitonin, a CCK or CCK agonist, a leptin or leptinagonist, an exendin or exendin analog agonist, a GLP-1 or GLP-1 analogagonist, a DPPIV inhibitor, or a PYY or PYY analog.
 27. The method ofclaim 26, wherein the exendin is exendin-3 or exendin-4.